Analyzing the Viral Resistance of Tomato Plant to TYLCV, PVY, and TSWV Under Heat and Drought Conditions

Analyzing the viral resistance of tomato plant to TYLCV, PVY, and TSWV under heat and drought conditions

Der Naturewissenschaftlichen Fakultät der Friedrich Alexander Universität Erlangen-Nürnberg Zur Erlangung des Doktorgrades Dr. rer. nat.

Vorgelegt von Wafa'a Odeh

Aus Jordanien

Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 20.02.2018 Vorsitzende/r des Promotionsorgans: Prof. Dr.Georg Kreimer. Gutachter/in: Prof. Dr. Uwe Sonnewald. Prof. Dr. Wolfgang Kreis.

To the soul of my mother

& To my beloved family

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Table of Contents Summary ...... 1 Zusammenfassung ...... 3 1. Introduction ...... 5 1.1 Tomato ...... 5 1.1.1 An important crop ...... 5 1.1.2 Tomato is a sensitive crop plant ...... 6 1.2 Viral diseases of tomato plants ...... 6 1.2.1 Tomato yellow leaf curl virus (TYLCV) ...... 8 1.2.2 Tomato spotted wilt virus (TSWV) ...... 9 1.2.3 Potato virus Y (PVY) ...... 10 1.3 Plants are exposed to different abiotic stress ...... 11 1.4 Climate change and abiotic stress ...... 12 1.5 Response of plants to combined biotic and abiotic stress ...... 12 1.6 Plant receptors: key role in pathogen recognition ...... 15 1.7 Host factors; an important role in viral pathogenicity ...... 17 1.8 Development of virus resistant plants ...... 20 1.9 Aims of this study ...... 21 2. Materials and methods ...... 23 2.1 Materials ...... 23 2.1.1 Chemicals, enzymes, and consumables ...... 23 2.1.2 Bacterial strains ...... 23 2.1.3 Antibiotics and additives ...... 23 2.1.4 Vectors ...... 24 2.1.5 Oligonucleotides and sequencing ...... 24 2.1.6 Media ...... 25 2.1.7 Plant materials ...... 26 2.1.8 Constructs for plant transformation ...... 26

2.2 Methods ...... 27 2.2.1 Growth conditions and transformation ...... 27 2.2.1.1 Growth conditions and transformation of Bacteria ...... 27 .2.21.2 Growth conditions and transformation of plants ...... 28 2.2.2 Transcriptome analysis of tomato plants exposed to triple stress (virus, heat, and drought) ...... 28 2.2.2.1 Field experiment in Jordan ...... 28 2.2.2.1.1 Sites selection and time frame ...... 28 2.2.2.1.2 Soil analysis ...... 28 2.2.2.1.3 TYLCV transfection of tomato plants ...... 28 2.2.2.1.4 Plants in the field ...... 29 .22.2.1.5 Temperature monitoring ...... 29 2.2.2.1.6 Monitoring the water content of the soil ...... 29 2.2.2.1.7 Sample collection ...... 30 2.2.2.1.8 Symptoms monitoring ...... 30 2.2.2.2 Transcriptome analysis ...... 30 2.2.2.2.1 RNA isolation ...... 30 2.2.2.2.2 Microarray hybridization ...... 31 2.2.2.2.3 Analysis of Microarray data ...... 31 2.2.2.2.4 DNA extraction, PCR amplification, and sequencing analysis31 2.2.2.2.5 Prediction of the secondary structure of the LRR-RLP ...... 32 2.2.2.2.6 Quantitative real-time RT-PCR (Q-RT-PCR) of the gene receptor-like kinase (RLK) ...... 32 .22.2.2.7 Analyzing the conserved domain of RLK and LRR-RLP ..... 32 2.2.3 Transformation of Nicotiana tabaccum and Solanum lycopersicum with constructs having single or double genes ...... 32 .2.23.1 Preparation of the construct with the single gene (NSm) ...... 32 2.2.3.2 Stable transformation of N. tabaccum and S. lycopersicum plants33 2.2.3.2.1 Stable transformation of tobacco and tomato plants ...... 33 2.2.3.2.2 Analyzing regenerated plantlets ...... 33 2.2.3.2.3 Testing transgenic N. tabaccum plants for TSWV resistance 34 2.2.3.3 Selection of homozygous transgenic lines ...... 34 2.2.4 Identification of host factors interacting with TYLCV ...... 35 2.2.4.1 DNA Isolation ...... 35

.2.24.2 Rolling circle amplification (RCA) of TYLCV DNA ...... 35 2.2.4.3 Sequencing of TYLCV isolates ...... 35 2.2.4.4 Cloning the open reading frames (ORFs) of TYLCV ...... 35 2.2.4.5 Subcellular Localization of TYLCV ORFs ...... 36 2.2.4.6 Immunoprecipitation of fused protein of V1, V2, and C2 ...... 36 2.2.4.7 Tryptic digestion of the precipitated protein ...... 37 2.2.4.8 Analysis of precipitated protein via Nano LC-Mass Spectrometry37 2.2.4.9 Data analysis ...... 38 3. Results ...... 39 3.1 Transcriptome analysis of tomato plants exposed to triple stress ...... 39 3.1.1 Temperature records at both sites ...... 40 3.1.2 Evaluation of disease symptoms ...... 41 3.1.3 Transcriptome analysis of field samples ...... 43 3.1.4 Differential gene regulation in resistant and susceptible lines ...... 46 3.1.5 Differentially regulated genes located on chromosome 11 ...... 48 3.1.6 Relative expression of Receptor-like kinase (RLK) ...... 52 3.1.7 Conserved domains of RLK and LRR-RLP ...... 53 3.2 Evaluation of Potato virus Y (PVY) and Tomato spotted wilt virus (TSWV) resistance in transgenic Nicotiana tabacum SNN and Solanum lycopersicum M82 lines having single or double gene constructs ...... 54 3.2.1 Tobacco and tomato plants expressing single or double genes ...... 54 3.2.2 TSWV resistance in tobacco plants transformed with the single or double genes ...... 58 3.2.3 T1 transgenic tobacco and tomato lines ...... 60 3.3 Interaction partners of Tomato yellow leaf curl virus (TYLCV) ...... 61 3.3.1 TYLCV working isolate ...... 61 3.3.2 Subcellular localization of V1, V2, and C2 ...... 62 3.3.3 Candidate interaction partners of TYLCV ORFs ...... 63 4. Discussion ...... 65 4.1 Gene expression does not respond to treatments ...... 65 4.2 LRR-RLP interacts with RLK to initiate signaling and mediates resistance response to TYLCV in resistant lines ...... 66 4.3 Resistant lines are distinguished from susceptible lines via SNPs detected in LRR-RLP ...... 68

4.4 A single amino acid change may alter the recognition specificity of the receptor like protein ...... 68 4.5 ∆M541 and NSm don´t confer tobacco plants resistance to TSWV ...... 69 4.6 TYLCV proteins interact with host factors ...... 70 5. List of abbreviation ...... 75 6. References ...... 77 7. Appendix ...... 92

8. Acknowledgement ...... 102

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Summary

Summary Tomato is a worldwide grown crop. Its growth and survival are influenced by pathogens and by climate change. Heat and drought resulting from climate change will greatly affect plant-pathogen interaction. Among pathogens that infect tomato, viruses like Tomato yellow leaf curl virus, Potato virus Y, and Tomato spotted wilt virus are of great importance as they are limiting tomato productivity.

In this work, multiple stresses (heat, drought, and virus) have been simultaneously applied to both TYLCV resistant (T3, F7) and susceptible (981) tomato plants to understand the molecular and physiological response of the tomato plant to these stresses. Transcriptome analysis showed that samples were not grouped according to the environmental conditions that have been applied and the clustering pattern showed that differently treated plants grouped together in the same cluster .

From the list of differentially expressed genes, thirteen entities were shown to be differentially up regulated in the resistant lines T3 and F7 when they were compared to the susceptible line (981), regardless of the conditions that plants were grown in (heat, drought) and whether these plants were TYLCV infected or healthy. Sequencing genomic DNA of three selected candidate genes, Erwinia induced protein 2, Receptor like kinase, and Splicing factor 3B subunit 4, from resistant and susceptible cultivars showed that the sequences amplified from TYLCV resistant lines (GF13 X 981 (F7), GF13 X 967 (F5), Favi-9) shared high sequence identity with those of the TYLCV susceptible lines (981, 967). Nine single nucleotide polymorphisms (SNPs) were detected when the nucleotide sequence of the LRR-RLP gene from resistant and susceptible lines was compared. One of the SNPs was observed in the cDNA sequence of LRR- RLP. The nucleotide adenine in position 382 of the cDNA of susceptible lines

Summary has been replaced by the nucleotide guanine in the resistant lines. The substitution of the nucleotide A382 to G382 leads to the amino acid change from arginine in susceptible lines to glycine in resistant lines. It is postulated that LRR-RLP in resistant lines interacts with RLK to mediate resistance response to TYLCV in resistant lines.

To understand the resistance of tomato to PVY and TSWV and its stability under unfavorable conditions (heat, drought), tobacco plants were generated expressing either the movement protein of TSWV or truncated, dominant- negative versions of the dnaJ proteins CPIP (ΔCPIP1) and M541 (ΔM541). To obtain resistance against both, potyviruses and Tospoviruses, single and double transformations were carried out. Unfortunately, none of the transgenic tobacco plants were resistant against TSWV.

Several host factors interacting with TYLCV proteins V1, V2, and C2 were identified using co-immunoprecipitation and mass spectrometry. It is postulated that the interacting host factors have a role in the infection cycle of TYLCV.

Zusammenfassung

Zusammenfassung Die Tomate ist eine weltweit häufig angebaute Nutzpflanze, deren Wachstum und Überleben bedingt durch den Klimawandel von einer immer größer werdenden Anzahl an Krankheitserregern bedroht wird. Hitze und Trockenheit sind Folgen des Klimawandels und können die Pflanzen-Pathogen Wechselwirkungen stark beeinflussen. Zu den Krankheitserreger die für signifikante wirtschaftliche Einbußen bei dem Anbau von Tomaten verantwortlich sind zählen unter anderem Viren wie zum Beispiel TYLCV, PVY und TSWV.

Um einen Einblick in die molekularen und physiologischen Antworten von Tomatenpflanzen auf multiple Stressfaktoren (Hitze, Trockenheit und Viren) zu erhalten, wurden TYLCV-resistente Tomatenpflanzen (T3, F7) und TYLCV- suszeptibele Tomatenpflanzen (981) gleichzeitig verschiedenen Stressbedingungen ausgesetzt. Eine anschließende Transkriptomanalyse zeigte, dass die verschiedenen Proben nicht hinsichtlich der ausgelieferten Umwelteinflüsse gruppierten.

Mittels Clusteranalysen konnten dreizehn Gene identifiziert werden, deren Expression im Vergleich zur Expression in den Tomatenpflanzen der Linie 981, spezifisch in den TYLCV-resistenten Tomatenlinien T3 und F7 nach oben reguliert waren. Diese Regulation war unabhängig von den Bedingungen, in denen die Pflanzen angezogen wurden (Hitze, Trockenheit) und unabhängig davon, ob die Pflanze gesund oder mit TYLCV infiziert waren. Die Sequenzanalyse der Kandidatengene (Erwinia induced protein 2, RLK, Splicing factor) zeigte, dass die aus TYLCV-resistenten Linien isolierten genomischen DNA-Sequenzen (GF13 X 981 (F7), GF13 X 967 (F5), Favi-9) eine hohe Sequenzidentität mit den Sequenzen, welche aus den TYLCV-empfänglichen Linien (981, 967) isoliert wurden, aufwiesen. Insgesamt konnten neun Einzelnukleotidpolymorphismen (SNPs) beim Vergleich der Nukleotidsequenz

Zusammenfassung des LRR-RLP-Gens zwischen den resistenten und den suszeptiblen Linien identifiziert werden. Einer der SNPs in dem LRR-RLP Gen konnte auch in einer aus cDNA amplifizierten Sequenz nachgewiesen werden. Bei den suszeptiblen Linien lag an Position 382 der Nukleotidsequenz ein Adenin vor, wohingegen bei den TYLCV-resistenten Linien ein Guanin an Position 382 kodiert war. Diese Substitution von A382 zu G382 führt zu einer Änderung der Aminosäure- Sequenz von Arginin (suszeptible Linien) hin zu Glycin in den TYLCV- resistenten Tomatenlinien. Nach allgemeiner Meinung interagiert LRR-RLP mit RLK um die Resistenzantwort gegenüber TYLCV in den resistenten Linien zu vermitteln.

Um die Resistenz der untersuchten Tomatenlinien gegen PVY und TSWV und das Aufrechterhalten dieser Resistenz auch unter ungünstigen Umweltbedingungen (Hitze, Trockenheit) verstehen zu können, sollten transgene Tabakpflanzen erzeugt werden, die entweder das TSWV Transportprotein (Nsm) oder eine verkürzte, dominant-negative Form der dnaJ protein CPIP (ΔCPIP1) und M541 (ΔM541) exprimierten. Um sowohl eine Potyvirus als auch eine Tospovirus Resistenz zu erhalten, wurden neben den Einfachtransformationen auch Doppeltransformationen durchgeführt. Leider war keine der erzeugten transgenen Tabakpflanzen resistent gegenüber TSWV.

Mehrere Wirtsfaktoren, die mit den TYLCV-Proteinen V1, V2 und C2 interagierten, konnten unter Verwendung von Co-Immunpräzipitation und Massenspektrometrie identifiziert werden. Es wird vorgeschlagen, dass die interagierenden Wirtsfaktoren eine Rolle im Infektionszyklus von TYLCV spielen.

Introduction

1. Introduction

1.1 Tomato

1.1.1 An important crop

Tomato (Solanum lycopersicum) is one of the widely grown vegetable crops all over the world, Statistics of Food and Agriculture Organization stated that 177,042,359 tonnes of tomato were produced in the world in 2016; Europe is the third largest producer (13.7%) after Asia (60.1 %) and Americas (14.7 %) in the same year. In Germany, the average production of tomato was 85,287 tonnes; while it was 837,342 tonnes in Jordan for the same year 2014.The map and pie graph (Figures 1 and 2) below represent the average tomato production in all countries all over the world and the share of each continent of worldwide production of tomato respectively (FAOSTAT, 2016).

Figure 1: Production quantities of tomatoes in tonnes by country for the year 2016. Dark red shaded counties are high tomato producers, lighter red shaded are low tomato producer countries (http://faostat.fao.org).

Introduction

Figure 2: Production share of tomatoes by region for the year 2016. Asia produces about two- third of tomato all over the world (http://faostat.fao.org). 1.1.2 Tomato is a sensitive crop plant

Unfortunately, the tomato is a target to a high number of diseases and pathogens that significantly affecting tomato production (Murad et al., 2009; Pradeep and Masato, 2010; Sead et al., 2009; Panthe and Chen, 2010). Tomato production and cultivation have been limited by more than 200 diseases caused by viruses, bacteria, fungi and nematodes (Lukyanenko, 1991; Jones et al., 1991; Aries et al., 2007; Cavalieri et al., 2014; Hanssen et al., 2010). In addition to biotic stress, many studies analyzed tomato plant response when exposed to both biotic and abiotic stresses simultaneously (Kissoudis et al., 2015; Moshe et al., 2012; Gorovits and Czosnek, 2008; Triky-Dotan et al., 2005).

1.2 Viral diseases of tomato plants Plant pathogens are an important factor in limiting agricultural productivity worldwide. Among plant pathogens, viruses are responsible for large yield losses (Verlaan et al., 2013). Brunt et al. (1996) reported that about 136 viral species could infect tomato crops. The number of viral species infecting other crops is much lower compared to tomato, for example, 44 viruses infect eggplant (S. melongena), 54 species infect potato (S. tuberosum), 46 that infect melon (Cucumis melo), 53 infect lettuce (Lactuca sativa), and 49 viruses infect pepper (Capsicum annum). The infection by plant viruses results in a wide range of symptoms in

Introduction tomato, symptoms, which reach from mild chlorosis to severe developmental changes and sometimes death of the infected plant (Bazzini et al., 2007; Kasschau et al., 2003). A large number of viruses are known to infect tomato plants all over the world, such as: Tomato yellow leaf curl virus (TYCLV; Genus Begomovirus) (Cohen and Harpaz 1964), Pelargonium zonate spot virus (PZSV; Genus Anulavirus) (Gallitelli 1982), Potato virus Y (PVY; Genus Potyvirus) (Jones et al., 1991), Tomato infectious chlorosis virus (TICV; Genus Crinivirus) (Duffus et al., 1996), Tomato chlorosis virus (ToCV; Genus Crinivirus) (Wisler et al., 1998), Tomato Spotted wilt virus (TSWV; Genus Tospovirus) (Peters D., 1998), Pepino mosaic virus (PepMV; Genus Potexvirus) (van der Vlugt et al,. 2000), Capsicum chlorosis virus (CaCV; Genus Tospovirus) (McMicheal et al., 2002), Tomato yellow ring virus (TYRV; Genus Tospovirus) ( Hassani-Mehraban et al., 2005), Tomato torrado virus (ToTV; Genus Torradovirus) (Verbeek et al., 2007), Tomato marchitez virus (ToMarV; Genus Torradovirus) (Verbeek et al., 2008), Tomato zonate spot virus (TZSV; Genus Tospovirus) (Dong et al., 2008), Tomato necrotic spot virus (TNSV; Genus Ilarvirus) (Batuman et al., 2009). Plant viruses that infect tomato are grouped into 33 genera; fifteen of them are of great economic importance, i.e. Alfalfamovirus, Begomovirus, Carlavirus, Crinivirus, Cucumovirus, Ilarvirus, Luteovirus, Nepovirus, Potexvirus, Potyvirus, Tobamovirus, Tombusvirus, Topocuvirus, Tospovirus, and Tymovirus (Petrov N., 2014). These genera belong to the following viral families Bromoviridae, Bunyaviridae, Closteroviridae, Flexiviridae, Geminiviridae, Luteoviridae and Potyviridae (Pringle, 1999). TYLCV has the most economical importance among viruses that infect tomato plants (Czosnek, 2007). TYLCV severely limits tomato production (Hanssen et al., 2010). The incidence of TYLCV may reach up to 100% in protected and open fields causing economic losses that could range from 50% to 90% of the crop production (Hamilton et al., 2015). TYLCV results in lower income for tomato crop producers and higher prices for consumers (Lapidot and Friedmann, 2002). TSWV is one of the most damaging pathogens of tomato and other vegetable crops (Roselló et al., 1996). TSWV epidemics caused losses in tomato around $8.8 millions in Georgia in the growing season 2000 (Riley and Pappu, 2004). Potyviridae represented by Potato virus Y (PVY) is the largest group of RNA plant viruses that are responsible for about 40% of all diseases of virus origin (Ghosh et al., 2002). PVY infects not only potato but also other crops including tomato, pepper, and tobacco (Shukla et al., 1994) and causes severe crop losses (Szemes et al., 2002).

Introduction

Based on the information above, it is concluded that TYLCV, TSWV, and PVY are viruses of great economic importance on tomato crop production worldwide, so these viruses will be introduced in details in the following section.

1.2.1 Tomato yellow leaf curl virus (TYLCV) Tomato yellow leaf curl virus is a geminivirus that belongs to the genus Begomovirus (Cohen and Harpaz 1964), the virus infects cultivated tomato in tropical and subtropical regions (Picó et al., 1996). The virus was first reported in the Middle East in the 1960s (Cohen and Hapaz, 1964), then the virus spread from the Middle East to different geographical regions (Lefeuvre et al., 2010). TYLCV is transmitted by the whitefly Bemisia tabaci (Glick et al., 2009; Kanakala and Ghanim, 2016). Symptoms caused by TYLCV include stunted growth, smaller leaf size, upward curling of the infected leaves accompanied with leaf mottling and chlorosis, and flower abscission, (Polston et al., 1999; Gilbertson et al., 2007). The genome of TYLCV is monopartite, circular, single-stranded DNA (Navot et al., 1991) that is about 2.8 Kb in size (Czosnek, 2007). Navot et al. (1991) reported that TYLCV genome has six partially overlapping open reading frames (ORF) that are organized into bidirectional transcriptional units separated by intergenic region (IR).

Figure 3: Genome organization of Tomato yellow leaf curl virus. The single stranded DNA is around 2.8 Kb in size (Czosnek, 2007). The genome contains six open reading frames. V1 and V2 are transcribed in a virion sense orientation, while C1, C2, C3, and C4 are transcribed as complementary sense strand. IR refers to the intergenic region. V1 encodes the capsid protein (CP); V2 is the movement protein; C1 the replication initiator protein (Rep); C2 is a transcription activator protein (TrAP); C3 a replication enhancer protein (REn); and C4 is a symptom determinant (Gronenborn, 2007).

Introduction

The ORFs V1 and V2 are transcribed on the viral sense strand, while the ORFs C1, C2, C3, and C4 are transcribed on the complementary strand (Gronenborn, 2007). Figure 3 shows the organization of TYLCV genome as reported by Gronenborn (2007). The intergenic region (IR) that is about 200 nucleotides contains promoters for transcription of the viral-sense genes (V1, V2) and the complementary sense genes (C1, C4) (Gronenborn, 2007), while The promoter for the transcription of the complementary sense genes C2 and C3 is located within the complementary gene C1 (Mullineaux et al., 1993). The ORF V1 encodes capsid protein (CP) that has functions in genome encapsidation, facilitating the movement of the genome in and out of the nucleus, transmission specificity of the whitefly vector, and it is required for the systemic infection of the plant (Díaz-Pendón, 2010). CP that localizes in the nucleus or it may be found associated with the nucleolus facilitate the movement of ssDNA and dsDNA and as they function as nuclear shuttle proteins of the bipartite begomoviruses (Rojas et al., 2001). V2 ORF encodes the movement protein, it is involved in viral movement and also it has a role in the suppression of gene silencing (Pakkianathan et al., 2015). C1 ORF encodes the replication initiator protein (Rep) that initiates the viral replication (Elmer et al., 1988). Rep protein has a nicking-closing activity; it initiates rolling circle replication by a site-specific cleavage in the viral replication origin (Lauf et al., 1995). The transcription activator protein (TrAP) is encoded by the ORF C2 and it localizes mainly in the nucleus of infected plant cells (Rosas-Díaz et al., 2015). C2 enhances the transcription of the viral genes (Sunter and Bisaro, 1991; 1992), and it has been shown to functions as a suppressor of RNA silencing (Trinks et al., 2005; Dong et al., 2003). The ORF3 that encodes the replication enhancer protein (REn) interacts with Rep protein for optimal replication of viral protein (Settlage et al., 2005). REn increases the DNA accumulation of geminiviruses (Elmer et al., 1988) and enhances symptom expression (Hormuzdi and Bisaro, 1995). The ORF C4 is known to be the symptoms determinant protein and also involved in silencing suppression (Pakkianathan et al., 2015).

1.2.2 Tomato spotted wilt virus (TSWV) TSWV belongs to the genus Tospovirus in the family Bunyaviridae (Milne and Francki, 1984). Tomato spotted wilt disease was first reported in 1919 in Australia (Wilson, 1998). TSWV has been common in Western Europe and USA (Aramburu and Marti, 2003), then the virus spread worldwide in temperate, tropical, and subtropical region (Mateus et al., 2012). Recently, TSWV was reported in the Middle East and far eastern Asia (Pappu et al.,

Introduction

2009). In Jordan, the first incidence of TSWV on tomato was reported by Anfoka et al., (2006). TSWV is transmitted efficiently by western flower thrips Frankliniella occidentalis (Kirk and Terry, 2003). Plants infected with TSWV are showing diverse symptoms including ring spots, black streaks on stem and petioles, apical bud death, and necrotic leaf spots (Roselló et al., 1996; Sherman et al., 1998). The genome of TSWV composed of three single-stranded RNA segments which are named base on their sizes: large (8.9kb), medium (4.8kb), and small (2.9kb) (Prins and Goldbach, 1998). The tripartite genome of TSWV is of negative/ambisense polarity (Kormelink et al., 2011). The schematic illustration of TSWV genome is presented in figure 4 as reported by Lee et al., (2011). The L RNA encodes the RNA-dependent RNA polymerase (RdRP) that has an important role in viral replication (de Haan et al., 1991). The M RNA encodes two genes; the nonstructural protein (NSm) which required for cell-to-cell movement, and the glycoprotein precursor (Gn-Gc) (Soellick et al., 2000). The nucleocapsid protein (N) and the nonstructural protein (NSs) which has a role in the suppression of gene silencing are encoded by S RNA (Bucher et al., 2003).

Figure 4: Genome organization of TSWV, the genome has three RNA segments: Large (L), medium (M), and small (S). Proteins of gray shading are translated in antisense orientation, while those shaded in white are translated in sense strand orientation (Lee et al., 2011). 1.2.3 Potato virus Y (PVY) PVY belongs to the genus Potyvirus, family Potyviridae (Walsh et al., 2001). Although 40 species of aphids belonging to 20 different genera can transmit the virus, the aphid Myzus persicae is the most efficient vector of PVY (Sigvald, 1984). PVY- infected tomato showing symptoms including vein yellowing, leaf mottling, mosaic, necrosis, leaf crinkling and dropping (Jones 1991; AVRDC Publication 2005).

Introduction

The genome of PVY consists of a single-stranded, positive-sense RNA molecule of about 10 kb in length, with a genome linked protein (VPg) attached covalently to the 5` end and a poly (A) tail at the 3` end (Figure 5) (Tribodet et al., 2005; Lorenzen et al., 2006). The virus replicates via double-stranded RNA intermediates (Missiou et al., 2004). The viral genome is expressed as a single polyprotein which is cleaved by three viral proteases P1, HC- Pro, and NIa into ten mature proteins (P1, HC-Pro, P3, 6 K1, CI, 6 K2, VPg, NIa-Pro, NIb, and CP) (Urcuqui-Inchima et al., 2001). An additional open reading frame named P3N-PIPO (Pretty Interesting Potyviridae ORF) and is translated +2 frameshift slippage of P3 has been found by Chung et al., (2008). Some studies suggested that P3 and P3N-PIPO have a role in the intercellular movement of potyviruses (Wei et al., 2010; Wen and Hajimorad, 2010).

Figure 5: Genome organization of PVY (Cuevas et al., 2012 modified). The viral genome consists of a single-stranded, positive sense RNA with a genome linked protein (VPg) (the dark green circle) attached covalently to the 5' end and a poly A tail ((A)n) at the 3' end (Tribodet et al., 2005). P1 is a proteinase, HC-Pro: helper component proteinase that is involved in aphid transmission and polyprotein processing (Shukla et al., 1994), P3: has a role in intercellular movement (Wei et al., 2010), 6K1 and 6K2 it is thought to have a role in genome replication (Shukla et al., 1994), CI: cylindrical inclusion protein is an RNA helicase and recently found to have a role in virus movement (Shukla et al., 1994; Wei et al., 2010), NIa: nuclear inclusion protein a is a proteinase, NIb: nuclear inclusion protein b is an RNA dependent RNA polymerase (RdRp) (Shukla et al., 1994), and CP has a role in RNA encapsidation, aphid transmission and cell-to-cell movement, VPg: involved in genome replication (Shukla et al., 1994: Rojas et al., 1997). 1.3 Plants are exposed to different abiotic stress Open field grown crops face variable conditions which limit plant growth and yield (Kissoudis et al., 2015). Abiotic stresses are important limiting factors that influence plant growth and crop production (Gorovits and Czosnak, 2008). Abiotic stress such as heat, cold, drought, salinity, and nutrient stress have a great effect on world agriculture, and it has been suggested that they reduce average yields by more than 50% for most major crop plants (Wang et al., 2003). A combination of more than one stress condition is a common to occur under normal conditions in many agricultural areas, this will magnify the effect and increase the losses caused by each stress alone (Suzuki et al., 2014).The current climate change

Introduction prediction indicates that the ambient temperature is gradually increasing, so the frequency and amplitude of heat stress is enhanced (Mittler and Blumwald, 2010). Under natural conditions, plants are facing the threat of infection by pathogens (bacteria, fungi, viruses, nematodes, or pests) in addition to abiotic stresses (Atkinson and Urwin, 2012). Stress (either biotic or abiotic) will reveal a complex of cellular and molecular response systems implemented by the plant in order to ensure survival (Pandey et al., 2015).

1.4 Climate change and abiotic stress Evidence for global climate warming is clear and now widely accepted (Solomon et al., 2007). Plant growth and survival, as well as the development of plant disease would be greatly affected by the drought and temperature stresses resulting from climate change (Colhoun, 1973, 1979). Resistance to pathogens like bacteria, fungi, virus, and insects is influenced by temperature (Garrett et al., 2006). Roggero P. (2002) reported that Tsw- mediated TSWV resistance is broken under elevated temperatures. Plant biologists are making great efforts to study the effect of climate change parameters on the disease epidemics and plant-pathogen interaction (Eastburn et al. 2011). Plant pathogen interactions are modulated by environmental factors that could affect the incidence and severity of plant disease, and affect also defence responses of the host (Browder 1985; Colhoun 1973; Eastburn et al., 2011). The development of any plant disease is a result of the interaction of a susceptible host, a virulent pathogen, and an environment that suits disease development (Eastburn et al. 2011).

1.5 Response of plants to combined biotic and abiotic stress Plants are adapted to different environmental conditions, they respond to them by activating specific molecular and physiological processes in order to survive under stress condition (Atkinson et al., 2013). Abiotic stress is responsible for growth reduction in most plant species (Wang et al., 2003). Moreover, biotic stress adds an extra pressure to the damaged plants (Maron and Crone, 2006; Brown and Hovmoller, 2002). Rhizky et al. (2002; 2004) reported that the molecular response of plants to a combination of stress is unique and cannot be extrapolated from plant response to each stress individually. The simultaneous exposure of plants to biotic and abiotic stress might have a negative or positive effect on the plants; the resulting effect depends on the interaction between the abiotic stress and the pathogen (Tippmann et al., 2006).

Introduction

Many studies indicated that the defence response of plants to pathogens is suppressed when they are exposed to the high temperature (Rmegowda et al., 2015; Sharma et al., 2007; Want et al., 2009). Kiraly et al., (2008) showed that high temperature increases the susceptibility of tobacco (Nicotiana tabacum) and pepper (Capsicum annum) to tobacco mosaic virus (TMV). In contrast, some studies indicated that plants showed resistance to the pathogen when they were exposed to abiotic stress simultaneously (Rmegowda et al., 2015). Atkinson and Urwin, (2012) reported that the severity of drought stress is decreased in rice plants that have been exposed to combined drought stress and nematode infection. Plants are adapted to stress by activating special changes in physiological, molecular, cellular process and signaling pathways to minimize the resulting damage (Ben Rajeb et al., 2014). An important step for plants to defend the stress is the perception and recognition of stress (Ben Rajeb et al., 2014). When abiotic and/ or biotic stress is recognized, specific ion channels and kinase cascades are activated (Fraire-Velázquez et al., 2011), reactive oxygen species (ROS) (Laloi et al., 2004), callose (Yasuda et al., 2008), and phytohormones such as abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA) and ethylene (ET) (Spoel and Dong, 2008) are accumulated, and the genetic profile of the plant is reprogrammed (Fujita et al., 2006). Reactive oxygen species (ROS) are signaling molecules that are rapidly generated by the plant following stress recognition to avoid tissue damage (Foyer and Noctor, 2005). Low levels of ROS are regulating plants' stress response, while high levels are destructive and causing cell death (Choudhury et al., 2013). Recognition of stress is followed by the activation of mitogen-activated protein kinases (MAPKs) that control the pathway of the stress response (Ben Rajeb et al., 2014; Zhang et al., 2006a; Wurzinger et al., 2011). Under different abiotic and biotic stress response, MAPKs are responsible for signal transduction of several cellular processes (Fujita et al., 2006). The activation of the MAPKs pathways is mediated by SA and that result in an expression of pathogenesis related (PR) proteins (Xiong and Yang, 2003). MAPK signaling also interacts with the signaling pathway of ROS and ABA (Miura and Tada, 2014; Zhou et al., 2014). Plant hormones play important roles not only in regulating developmental processes but also have a crucial role in signaling networks involved when a plant respond to biotic and abiotic stresses (Bari and Jones, 2009). Auxins, gibberellins (GA), cytokinins (CK), ABA, ET, SA, JA, brassinosteroids (BR) and strigolactones are major hormones produced by the plant (Verma et al., 2016). ABA, SA, JA, and ET are known to control plant defence response against pathogen and abiotic stress (Bari and Jones, 2009; Nakashima and Yamaguchi-

Introduction

Shinozaki, 2013). ABA is known to be responsible for plant response against abiotic stress since ABA levels are increased when plants are exposed to drought, cold, salinity, and heat stress (Lata and Prasad, 2011; Zhang et al., 2006b). On the other hand, SA, JA, and ET are involved in plant response to biotic stress as the pathogen infection leads to an increased level of these hormones (Bari and Jones, 2009). Nishiyama et al., 2011 reported that plant defence response is not restricted to ABA, SA, JA, and ET, other hormones such as auxins, GAs, and CKs are interacting directly or indirectly with other hormones to help plants to adapt to any stress.

Figure 6: Elements involved in the regulation of biotic and abiotic stress in plants. The first step is that the biotic and abiotic stresses have been recognized by sensors, and then the message should be transduced to downstream pathways. Reactive oxygen species (ROS) and Calcium ions (Ca+2) are second messengers. ROS and Ca+2 activate mitogen-activated protein kinases (MAPK). ROS will affect abscisic acid (ABA) availability in the cytoplasm. ABA signaling is mainly involved in abiotic stress adaptation; it is responsible to induce callose deposition. ABA is reacting positively with JA/ET signaling. SA activation by a pathogen will attenuate ABA responses. ABA has a negative effect on the signals that trigger systemic acquired resistance; this helps the pathogen to spread from the site of infection. The interaction of SA, JA, and ET signaling will increase pathogen resistance of the plant. Hormones (ABA, SA, JA, ET) , priming agent, and secondary metabolites (flavonoids, polyamines, Brassinosteroid) and other cytoplasmic chemicals will up-regulate the defence genes, transcription factors (TR) pathogenesis related proteins (PR), the genes of heat shock proteins (HSP) and other genes that have a role in protecting the cell against stress. Arrows indicate induction while flat-ended lines indicate repression.

Introduction

One of the important events that take place after stress detection is the change in gene expression (Ben Rajab et al., 2014). The Change in the expression of specific genes is crucial for the plant to reach an effective defensive state since there is evidence that several genes are multifunctional and able to improve plants tolerance to stresses (Nakashima et al., 2009; Swindell, 2006; Sharma et al., 2013). The activity of genes that have a role in plants' defence is controlled by specific phytohormones such as ABA, JA, SA, and Ethylene (Mengiste et al., 2003). Many PR genes are induced when the plants are exposed to abiotic stress or when they are attacked by a pathogen; PR genes are highly up-regulated when plants are under stress (Seo et al., 2010). Figure 6 summarizes some pathways that have been activated when a plant is exposed to biotic and abiotic stress.

1.6 Plant receptors: key role in pathogen recognition Plants are immobile organisms that depend on innate immune responses to defend themselves against pathogens; every plant cell has the potential to trigger an immune response as they lack specialized immune cells or organs (Zipfel, 2014). Instead of specialized immune cell or organs, plants employ two layers innate immune system that includes cell surface localized and intracellular receptors (Dangl et al., 2013). Receptors perceive signals from endogenous and exogenous stimuli (Tör et al., 2009). Endogenous signals which derived from damaged or impaired cells are called damage-associated molecular pattern molecules (DAMPs) (Lotze et al., 2007). Exogenous signals might be (i) pathogen- or microbe- associated molecular pattern molecules (PAMP or MAMP) or (ii) non-microbial or abiotic stress inducers like pollutants, toxic compounds, injury, or ozone (Tör et al., 2009). Following stimuli detection, receptors have a significant role in the activation of pathways controlling defence and development (Tör et al., 2009). Those receptors are currently known as Pattern Recognition Receptors (PRRs) (Tör et al., 2009; Zipfel, 2014). Tör et al. (2009) have categorized receptors according to the presence and order of protein domains and the localization of the receptor as it is demonstrated in figure 7. The intracellular PRRs are nucleotide binding site-leucine-rich repeats proteins (NB-LRR); NB-LRR proteins either encode an N-terminal domain with Toll/Interleukin-1 Receptor homology (TIR-NBS- LRR), or N-terminal with a coiled-coil motif (CC-NBS-LRR) (Mayers et al., 2003). NB-LRR receptors are encoded by disease resistance genes (Mayers et al., 2003) and localized in the cytoplasm, but they could be translocated into the nucleus, chloroplast or mitochondria (Tör et al., 2009).

Introduction

Figure 7: Domain organization of typical extracellular and intracellular receptors in plants. Internal receptors, NB-LRR proteins recognize specific signals like effector molecules. They trigger signaling cascades and induce plant resistance to pathogens. Extracellular PGIP, RLP, and RLK-type receptors are categorized according to the extracellular domain. These receptors interact directly or indirectly with MAMPs and DAMPs signals in order to be recognized. Abbreviations: PGIP, polygalacturonase-inhibitor protein; RLP, receptor-like protein RLK, receptor-like kinase; LRR, leucine-rich repeats; LysM, lysine motif; PR5K, pathogenesis related 5-like receptor kinase; RLCK, receptor-like cytoplasmic kinase; S-domain, self- incompatibility domain; TNFR, tumour necrosis factor receptor; WAK, wall associated kinase; NB, nucleotide binding; TIR, Toll and Interleukin 1 transmembrane receptor; CC, coiled-coil; EGF, epidermal growth factor; RCC, regulator of chromosome condensation; Ser/Thr, serine/threonine. Extracellular PRRs are classified into the following (i) Receptor-like kinases (RLK) (Shiu and Bleecker, 2003); all RLKs shared a single transmembrane region and a cytoplasmic kinase domain, but they are distinguished by their extracellular domain. The extracellular domain has been used to classify RLKs into subfamilies including LRRs, lectin, lysine motif (LysM), wall associated kinase (WAK), self-incompatibility locus (S-Locus), tumor necrosis factor receptor (TNFR), PR5-like receptors (PR5K), and receptor-like cytoplasmic kinases (RLCKs) have been classified as RLKs although they don't have extracellular domains (ii) receptor-like proteins (RLPs) (Wang et al., 2008); typically possess an extracellular LRR domain and short C-terminal membrane anchor but lacking cytoplasmic kinase domain. (iii) Polygalacturonase-inhibiting proteins (PGIPs) (Di Matteo et al., 2003) have only an extracellular LRR domain.

Introduction

Many RLPs have been identified in different plant species. In Arabidopsis, 56 RLPs have been identified, while more than 90 RLPs were identified in rice (Fritz-Laylin et al., 2005). These proteins play a crucial role in defence as well as in development, e. g. the Arabidopsis CLAVATA2 (CLV2: AtRLP10) and Too Many Mouths (TMM: AtRLP17) regulate meristem and stomata development respectively (Jeong et al., 1999; Shpak et al., 2005). Andolfo et al. (2013) revealed the distribution of plant receptors all over tomato genome, about 173 RLPs and 256 RLKs are distributed unevenly on tomato chromosomes. RLPs Cf (2, 4, 4E, 5, 9B) are involved in the resistance to Cladosporium fulvum (Dixon et al., 1996; Takken et al., 1999; Panter et al., 2002; Rooney et al., 2005; Liebrand et al., 2013). RLP Ve is responsible for tomato resistance to Verticillium species (Kawchuk et al., 2001). A total of 769 pathogen recognition genes in tomato were classified by Andolfo et al., (2013) according to the presence and order of protein domain, phylogenetic analysis and physical arrangement within the genome. Five typical pathogen recognition genes families (TIR-NBS-LRRs, CC-NBS-LRRs, RLKs, RLPs, and kinase-like proteins) were physically localized on the chromosomes of the tomato genome. It is thought that the defence response is the main biological function of the transmembrane receptors RLKs and RLPs. Phylogenetic analyses performed by Fritz-Laylin et al. (2005) could predict the candidate disease resistance receptor genes of Arabidopsis and rice. But for some plant species, it is still unclear which receptor genes are responsible for specific disease resistance, although a lot of studies have been accomplished to identify these disease resistance receptor genes (Andolfo et al., 2013). Since RLPs lack the cytoplasmic kinase domain, RLPs rely on RLKs to internalize the signal (Zipfel, 2014). Some studies showed that the RLPs CLVs and TMM are able to work functionally in combination with the RLKs CLAVATA1 and ERECTA respectively (Shpak et al., 2005; Waites and Simon, 2000).

1.7 Host factors; an important role in viral pathogenicity Viruses as obligate intracellular parasites are fully dependent on host machinery; this requires that the viral proteins and nucleic acids change the normal function of some host factors to serve the complex viral interactions required for the infection cycle (virus translation and replication, virus movement, viral pathogenicity and host response) (Hull, 2002). The identification of specific cellular factors that contribute to a viral pathogenicity is achieved through developing host model system, host genetics, functional genomics, and other molecular, biochemical, and biological methods such as yeast two-hybrid interaction (Whitham and Wang, 2004).

Introduction

The eukaryotic translation initiation factor (eIF4F) is associated with potyvirus susceptibility; eIF4F might interact with viral genome -linked protein VPg at the 5' end of the potyviral genome (Dunoyer et al., 2004). VPg interacts with eIF4E and its isoforms to support the translation of some potyviral RNAs, and it facilitates RNA replication and cell-to-cell movement (Wang and Krishnaswamy2012; Jing and Laliberte, 2011). Sanfaçon, 2015 suggested that mutations in eIF4E and its isoforms will provide broad-spectrum recessive resistance to PVY isolates. Cellular chaperones are important host factors for virus replication and cell-to-cell movement (Verchot, 2012). Heat shock proteins (HSP) 40, 60, 90, and 100 chaperones control the folding and aggregation of proteins (Mayer, 2010; Tyedmers et al., 2010). HSP70 and HSP90 are known to play a crucial role in the formation of membrane-bound replication complex for some members of tombusvirus, tobamovirus, potyvirus, and potexvirus. The co- chaperone J-domain protein (HSP40) directly interacts with HSP70 by recruiting the substrate to HSP70 by an ATP-dependent process (Verchot, 2012). Dna-j like protein (HSP40) was found to facilitate the movement function of PVY and TSWV by interacting with capsid protein (CP) of PVY and the movement protein (NSm) of TSWV (Hofius et al., 2007; Soellick et al., 2000). The viral genome of potyviruses do not encode a specialized movement protein, the movement is a function of other virus encoded proteins in addition to their role in viral infection such as CP (Rojas et al., 1997), CI (Carrington et al., 1998), P3 and P3N-PIPO (Wei et al., 2010), and HC-Pro protein (Shen et al., 2010) . Yeast-two-hybrid system has been performed to screen the host factors which interact with PVY CP in Nicotiana tabaccum, the Dna-j like proteins that were found as PVY CP interaction partner are designated as NtCPIPs by Hofius et al., (2007). A Dna-j like protein designated as NtDnaJ-M541 was found to interact with TSWV NSm when NSm was used as bait in yeast-two-hybrid system (Soellick et al., 2000). Hafren et al., (2012) has proposed a model describing how CPIP with the help of HSP70 are regulating the virion assembly and disassembly by direct or indirect interaction with CP. As the genome of potyviruses starts to replicate, the viral proteins including CP are continuously synthesized. CP will activate virion assembly process that suppresses the replication and translation. The CPIP binds to CP and delivers it to HSP70 to perform ubiquitination and degradation of CP and thus preventing the accumulation of CP. This HSP70 dependent mechanism suppression of CP accumulation will enhance the potyviral genome translation. Regarding NtDnaJ-M541, the exact role is not known, but it is postulated

Introduction that it is either HSP70 mediated virus movement or NtDnaJ-M541 is a motive force for the translocation of ribonucleoprotein to the plasmodesmata (Soellick et al., 2000). The cell-to-cell movement and the viral genome trafficking are achieved by the corporation of host cytoskeleton and membranes (Morozov and Solovyev, 2003). Wei et al., (2010) proposed that P3N-PIPO interacts with CI to form plasmodesmata-associated conical structures that are essential for the cell-to-cell movement of potyviruses. P3N-PIPO is found to interact with host protein PCaP1 (cation-binding protein) that localized to PD, the interaction of P3-PIPO-PCaP1 facilitate the intercellular transport of potyviral virions via PD (Vijayapalani et al., 2012). Moshe et al., (2015) indicated that as TYLCV is spreading in the infected plants, CP and V2 aggregates that contain viral and host proteins are formed, the formation and stability of these aggregates are dependent on the integrity of actin and microtubule component of the cytoskeleton. For the replication process of geminiviruses which TYLCV belongs to, host DNA polymerase and other factors are required for viral DNA synthesis during elongation step of viral replication (Hanley-Bowdoin et al., 2004). Viral replicase (Rep: also called C1) is responsible for the initiation and termination steps of rolling circle replication of geminiviruses (Laufs et al., 1995). Viral replisome is recruited and assembled by Rep protein of geminiviruses; replisome consists of viral proteins and host factors that have a role in DNA replication, repair, and other nuclear functions (Hanley-Bowdoin et al., 2013). The Replication enhancer protein (REn: also called C3) that enhances DNA accumulation and interacts with Rep and other host factors that involved in viral replication is probably a part of viral replisome (Settlage et al., 2005; Hanley-Bowdoin et al., 2013). Rep and REn bind to proliferating cell nuclear antigen (PCNA) that interact with many proteins that play a role in cell cycle regulation, DNA replication, and DNA repair (Castillo et al., 2003; Bagewadi et al., 2004). Rep protein of geminiviruses also interacts and binds to many host proteins such as: large subunit of the replication factor C complex (Luque et al., 2002), Histone H3 (Kong and Hanley-Bowdoin, 2002), mitotic kinesin (Kong and Hanley-Bowdoin, 2002), and minichromosome maintenance complex 2 (MCMC2) (Suyal et al., 2013). Götz et al. (2012) indicated the interaction between TYLCV proteins and heat shock proteins 70 (HSP70) from the TYLCV insect vector Bemisia tabaci, this interaction was proofed via microarray, real time PCR, and western blot analysis. Gorovitz et al., (2013a) reported that TYLCV CP exploits host plant HSP70 for several processes like viral translocation to and from the plant nuclei, genome packaging and virion assembly, intracellular movement of CP, and long distance movement of TYLCV.

Introduction

1.8 Development of virus resistant plants Plant breeding is an efficient approach to achieve virus resistance in plants, but it is a time-consuming and costly process (Lecoq et al., 2004). The development of virus resistant cultivars producing high yield and having excellent traits is achieved through several consequent steps (i) identifying the source of resistance by screening germplasm collection, (ii) determining the mode of resistance and identification of genetic markers for marker- assisted selection (MAS), (iii) introgression of resistant trait into superior cultivar and evaluating the performance of the new cultivar in case of a pathogen challenge (Kumar, 1999). Breeding programs that aimed to produce TYLCV resistant tomato cultivars started in the 1960s, the program focused on introgressing resistant trait found in wild tomato species into a domesticated tomato (Vidavsky and Czosnek, 1998). Five TYLCV resistance loci have been mapped from wild tomato species; the dominant genes Ty-1, Ty-2, Ty-3, and Ty-4, and one recessive gene ty-5 (Zamir et al., 1994; Hanson et al., 2000; Ji et al., 2007; Anbinder et al., 2009). Ty-1 and Ty-3 are mapped in closed positions to tomato chromosome 6 of the wild tomato Solanum chilense (Ji et al., 2007), Ty-4 is also derived from the wild tomato S. chilense but it is mapped to chromosome 3 (Ji et al., 2008). The dominant Ty-2 gene is derived from S. habrochites f glabratum accession B6013 (Kallo and Banerjee, 1990; Ji et al., 2009) and mapped to chromosome 11 (Hanson et al., 2000). The recessive ty-5 gene is derived from S. peruvianum and mapped to chromosome 4 (Anbinder et al., 2009). Engineered resistance is another approach that has been used to develop virus resistant crops in the past few decades: Important types of engineered resistance are: pathogen-derived resistance (PDR) and post transcriptional gene silencing (Goldbach et al., 2003). The concept of PDR was first identified by Sanford and Johnson (1985) as inserting sequences from a pathogen to the host to protect it from the pathogen. PDR can exploit the sequences of viral coat protein (CP), movement protein (MP), and replicase genes to provide resistance against some viruses (Baulcombe, 1996). The use of sequences of viral CP to achieve CP-mediated resistance (CPMR) has been the most widely used and showed a high level of resistance for a number of RNA viruses such as TMV, PVX, CMV, and TRV (Beachy, 1994; Baulcombe, 1996).The principle of CPMR relies on preventing the disassembly of the infecting virus due to the accumulation of the transgenic CP (Goldbach et al., 2003). For MP- mediated resistance (MPMR), it was successful when a sequence of mutagenized MP was used to achieve MPMR (Lapidot et al., 1993).

Introduction

PTGS (known also as RNA silencing) is a post transcriptional regulation of gene expression which involves degradation of nucleic acid through a specific RNA degradation mechanism (Goldbach et al., 2003; Baulcombe, 2004).The mechanism of PTGS involves specific degradation of transgene mRNA or target RNA, that has either the same or complementary sequences (Waterhouse et al., 1998). For viral resistance, a transgene consists of viral sequences will prevent the accumulation of viral RNA containing theses sequences (Stam et al., 1997). PTGS is triggered in plants by double stranded RNA (dsRNA) molecules that might be produced as replication intermediate for some RNA viruses, single stranded RNA (ssRNA) from a transgene that is copied into dsRNA by the host encoded RNA-dependent RNA polymerase, or inverted repeat transgene that directly produced dsRNA (Meins, 2000; Voinnet, 2001; Goldbach et al., 2003). Dicer-like dsRNase enzymes will break down dsRNAs into short interfering RNAs (siRNA) of 21-23 nt long, siRNAs then guide RNA degradation by RNA-induced silencing complex (RISC) that breaks down a complementary or target mRNA (Elbashir et al. 2001; Waterhouse et al., 2001). Abhary et al., (2006) made use of PTGS approach to develop transgenic tomato plants resistant to TYLCV, conserved non-coding regions from three isolates of TYLCV have been selected to prepare the silencing triggering construct. Glaves et al., (2014) indicated that RNA silencing can achieve resistance against different viruses by using different types of interfering RNA molecules such as (I) sense RNA, (II) antisense RNA, (III) inverted repeat (IR) sequences, or (IV) micro RNA (miRNA).

1.9 Aims of this study Among pathogens that infect tomato, viruses are considered a limiting factor for tomato production; TYLCV, PVY, and TSWV are viruses of great economic impact on cultivated tomato. A conventional method like breeding was a method of choice for a long time to obtain virus-resistant tomato. The advanced knowledge of molecular genetics of the viruses and hosts, more understanding of plant defence mechanisms, and understanding the biochemistry of virus-host interaction lead to the development of novel strategies to confer a plant an engineered resistance to viruses and to improve plant response when the plant is exposed to a viral challenge (Machado et al., 2017; Pandey et al., 2015; Mandadi and Scholthof, 2013; Rodrigues et al., 2009). Either using conventional or novel methods in developing virus-resistant crop, it is of major importance to develop a crop with durable resistance to viruses under favorable and

Introduction stressful environmental conditions. To reveal more facts about plant resistance against viruses, this study aimed to: - Analyze the gene expression profile in TYLCV- resistant and TYLCV-susceptible tomato plants grown under different environmental conditions in an open field experiment. - Develop transgenic tomato plants showing resistance to PVY and TSWV individually or combined, and to evaluate their performance upon a challenge of the target virus. - Identify host factors that play a role in TYLCV infection cycle.

Materials and methods

2. Materials and methods

2.1 Materials

2.1.1 Chemicals, enzymes, and consumables Unless stated otherwise, chemicals, enzymes, and consumables were purchased from Carl Roth GmbH (Karlsruhe), Sigma-Aldrich (St. Louis, USA), Fermentas (St. Leon-Rot), Roche Diagnostics GmbH (Mannheim), and Promega (Madison, USA). Kits for clean-up of plasmids, PCR products, and DNA fragments were obtained from Qiagen (Hilden).

2.1.2 Bacterial strains Bacterial strains that have been used for cloning procedures and plant transformation are listed in table 1.

Table 1: bacterial strains used in this study. Strain Genotype Reference E. coli XL1 Blue recA1 endA1 gyrA96 thi-1 hsdR17 Bullock et al. (1987) supE44 relA1 lac [F´proAB lacIq ZΔ M15 Tn10 (TetR)] E. coli DH5a F-/ endA1 hsdR17 (rK-, mK+) glnV44 Woodcock et al., 1989 thi-1 recA1 gyrA (Nalr) relA1 D Invitrogen (Karlsruhe) (lacIZYA-argF) U169deoR (F80dlacD(lacZ)M15) R Agrobacterium Rif ; with helper plasmid pGV2260 van Larebeke et al. C58C1 AmpR (Deblaere et al., 1985) (1974)

R: antibiotic resistance

2.1.3 Antibiotics and additives The following antibiotic concentrations were used for bacteria selection: ampicillin: 100µg/ ml, kanamycin: 50 µg/ ml, rifampicin: 50 µg/ ml, spectinomycin: 50 µg/ ml, streptomycin: 20 µg/ ml. For blue/ white screening, Isopropyl β-D-1-thiogalactopyranoside (IPTG) and 5-Bromo-4- chloro-3-indolyl-β-Dgalactosid (X-Gal) were added to a final concentration of 40 µM IPTG and 40 µg/ ml X-Gal. All antibiotics and additives were dissolved in water and sterile-filtered, except rifampicin and X-Gal which were dissolved in dimethyl sulfoxide (DMSO) and dimethylformamide (DMF), respectively. Media were autoclaved, and then antibiotics and then other components have been added.

Materials and methods

2.1.4 Vectors Vectors used for general cloning procedures and plant transformation are listed in the following table. Table 2: vectors used in this study. Vector Use and resistance Source/ Reference pCR-Blunt E. coli cloning vector, KanR Invitrogen (Karlsbad, USA) pGEM-T® Easy E. coli cloning vector, Promega (Madison, USA) AmpR pENTR/ D-TOPO® E. coli cloning vector, KanR Invitrogen (Karlsbad, USA) pK7FWG2 binary vector, SmR/ SpR Invitrogen (Karlsbad, USA) pK7WGF2 binary vector, SmR/ SpR Invitrogen (Karlsbad, USA) pRB-35S binary vector, SmR/ SpR Prof. Frederik Börnke pGWB661 binary vector, SpR Nakagawa et al., 2007

2.1.5 Oligonucleotides and sequencing All oligonucleotides used in this study have been ordered from Metabion (Martinsried) are listed in Table 3. Sequencing of plasmids and PCR fragments was done by GATC Biotech AG (Konstanz). Table 3: Primers used in this study. Primer Name Targrt Gene Primer sequence Q-RLK-F RLK GCGATCGTAGCACAGAATCA Q-RLK-R RLK GCGGCCGGTGAAATCACTAA CaEF1α-2_5’ Elongation factor 1α CCCTCAGACAAGCCACTCC CaEF1α-2_3’ Elongation factor 1α ACACGACCAACAGGCACAG Erw1-20 F Erwinia induced ATGGCGTTGGAATGGGTTGT protein2 Erw374-393 R Erwinia induced TCAATCCGAAGCCTTCATCT protein2 RLK E2E4 F RLK GGGCTGTTGATGCAGCGTGT RLK E2E4 R RLK ACCGGGCCTCTCAGCTCAGTTG RLK E7E8 F RLK TCAGTGGAAGGAGGAGCAACGA RLK E7E8 R RLK TGGATGCGTCGAATGCTTTACTC LRR 1-21F LRR-RLP ATGTGGGCATGTATCAAGTTC LRR 780-800R LRR-RLP ATACTATCAGCATCACCGACA LRR3,239- LRR-RLP GAAGGAGAGGTTGTGAATGT 3,258F LRR4,204- LRR-RLP TCAACCACATTTGGCTAATGTTC 4,046R SF-F 1-21 Splicing factor ATGGCGGAGAACCCTAATTGC SF-R 852-878 Splicing factor TGCAAATTTTAATGTTCTATTGTAGAG UH 234 F ΔCPIP1 GGATCCAACAATGGATGAGGCGTTGAAATCG UH 235 R ΔCPIP1 GTCGACTTAGTCAACAGTCCTGCCCAGCAC UH 236 F ΔM541 GGATCCAACAATGGAAGACGGGCTGAAGGGTG UH 209 R ΔM541 and NSm GTCGACTTATGCGTAGTCTGGCACATCATAAG WO1 F NSm GGATCCAACAATGTTGACTCTTTTCGGTAACAAG

Materials and methods

TY-FWP1 TYLCV sequencing GGCCCTATGGAAACAGTCCA TY-RWP1 TYLCV sequencing CAGCCTCAGACTGGTCGTTTC TY-FWP2 TYLCV sequencing TCTGCTATCACCCTCAATGAT TY-RWP2 TYLCV sequencing ATGGCCGCTTTAATGATTTG TY-FWP3 TYLCV sequencing CAGTACCGCAACCGTGAAGAAT TY-RWP3 TYLCV sequencing CCATTAACAATGTAATCAGAGC TY-V1 F TYLCV ORF V1 CACCACAATGTCGAAGCGACCAGGC TY-V1 R TYLCV ORF V1 ATTTGATATTGAATCATAGAAATAGATGCG TY-V2 F TYLCV ORF V2 CACCATGTGGGACCCACTTCTA TY-V2 R TYLCV ORF V2 GGGCTTCGATACATTCTGTATATTC TY-C2 F TYLCV ORF C2 CACCATGCAACCTTCGTCACCC TY-C2 R TYLCV ORF C2 AATACTCTTAAGAAACGACCAGTCT

2.1.6 Media LB and YEB media were used for the transformation of E.coli and Agrobacterium, respectively, while MS medium was used for plant transformation. List of media and their components are listed in the following table. Table 5: media used in this study. Medium Components LB-Medium (Solid) 0,5% (w/v) Yeast extract 1% (w/v) NaCl 1% Tryptone 1.5% (w/v ) Agar YEB-Medium (Solid) 0,5% (w/v) Bacto Beef extract 0,1% Yeast extract 1% Bacto-Trypton 0,5% Sacchrose 2mM Magnesium Sulfate 1.5% (w/v ) Agar MS medium (Tobacco) For one liter: 4,4 g MS medium including vitamins (M0222) (Duchefa, Netherland) 2g Sacchrose* 1ml Myoinositol* (100mg/ml) 8g Phytoagar ((Duchefa, Netherland) pH 5,75 MS Medium (Tomato) For one liter: 4,4g MS medium including vitamins (M0245) (Duchefa, Netherland) 2g Sacchrose* 1ml Myoinositol* (100mg/ml) 8g Phytoagar ((Duchefa, Netherland) pH 5,9 * These components are added after autoclaving

Materials and methods

2.1.7 Plant materials • Tomato lines that have been used in this study were provided from Prof. Ghandi Anfoka (Al-Balqaa Applied University, Al-Salt, Jordan). Tomato lines have been used are listed below: -TYLCV resistant transgenic line GF13 X 981 (T3) -TYLCV resistant breeding line GF13 X 981 (F7) -TYLCV susceptible line 981 -TYLCV resistant breeding line GF13 X 967 (F5) -TYLCV susceptible line 967 -TYLCV resistant line Favi-9 (TYLCV resistant parent used in the breeding program of GF13 X 981 (F7) and GF13 X 967 (F5)). TYLCV resistant breeding lines GF13X981 (F7) and GF13X967 (F5) were developed during a long project where the source of resistance was introduced to these lines by crossing 981 and 967 respectively with TYLCV resistant tomato breeding line called GF13 (http://www.plantpath.wisc.edu/GeminivirusResistantTomatoes/CDR/Mar03/Field03.htm) from Guatemala. These lines were developed over eight years through a regional research project funded by USAID (United States Agency for International Development) and the data are not published. TYLCV resistant transgenic line GF13 X 981 (T3) was obtained by Agrobacterium mediated transformation of the breeding line GF13X 981 with TYLCV silencing triggering construct designed by Abhary et al., (2006) according to the protocol developed by Hussain et al., (2008). • For plant transformation, Nicotiana tabaccum cultivar SNN and Solanum lycopersicum cultivar M82 were used in this study.

2.1.8 Constructs for plant transformation Five constructs have used for plant transformation in this study. The constructs ΔCPIP1-NSm and ΔCPIP1-ΔM541 contain two synthetic genes that have been designed to confer plants resistance to PVY and TSWV. The double gene constructs (designed by Ursula Hoja) were synthetically prepared by Geneart (Regensburg) and then used as a template to generate the three constructs containing the single genes (ΔCPIP1, ΔM541, NSm). Table 4 below summarizes constructs structure and detailed information.

Materials and methods

Table 4: the structure of the constructs used to transform tobacco and tomato plants. CaMV35S: 35S promoter of Cauliflower mosaic virus, OCS: octopine synthase terminator, ΔCPIP1: interaction partner of PVY CP without 68 amino acids from N-terminus, NSm: the movement protein of TSWV, ΔM541: the interaction partner of TSWV movement protein without 70 amino acids from N-terminus, HL: hyperlinker (29 aa), HA: Human influenza hemagglutinin tag. All cloning procedures were made through BamHI/SalI. Construct Construct structure Resistance Name HA ΔCPIP1- PVY, NSm CaMV35 DCPIP1 HL NSm OC TSWV S S 26k 3k 35k D D D HA

ΔCPIP1- CaMV35S DCPIP1 HL DM541 OCS PVY, ΔM541 TSWV 26kD 3kD 30kD

ΔCPIP1 CaMV35S DCPIP1 OCS PVY

26kD HA CaMV35S DM541 OCS ΔM541 TSWV

30kD

HA NSm NSm CaMV35 OC TSWV S S 35kD

2.2 Methods

2.2.1 Growth conditions and transformation

2.2.1.1 Growth conditions and transformation of Bacteria E. coli bacteria were incubated at 37° C in liquid or on solid LB medium containing appropriate antibiotics. Agrobacterium tumefaciens were grown at 28° C in liquid or on solid YEB medium supplemented with suitable antibiotics. Transformation of E.coli was carried out via standard heat shock protocol, or manufacturer’s protocols for purchased competent cells. Positive transformants were selected by adding the appropriate antibiotic to the medium. Transformation of Agrobacterium tumefaciens was carried out according to protocol by Höfgen and Willmitzer (1990).

Materials and methods

2.2.1.2 Growth conditions and transformation of plants Agrobacterium tumefaciens-mediated gene transfer was used for tobacco transformation as described by Rosahl et al. (1987) and to transform tomato according to Knapp et al., 1994. Transformed tobacco and tomato plants were grown under the following conditions: -Temperature; day temperature 21o C, night temperature 19o C.

- Photoperiod of 16 hours.

2.2.2 Transcriptome analysis of tomato plants exposed to triple stress (virus, heat, and drought)

2.2.2.1 Field experiment in Jordan

2.2.2.1.1 Sites selection and time frame This experiment has been carried out in summer season (May -August) of the year 2012 in Jordan. Two locations have been selected to conduct the experiment:

1- The campus of Al-Balqa Applied University (University, GPS coordinates: 32° 1' 26" N / 35° 43' 0" E 2- The Research Station of Al-Balqa Applied University (Homra, GPS: coordinates: 32° 05′ 06″ N/ 35° 38′ 52″ E).

2.2.2.1.2 Soil analysis Soil samples were collected from both selected sites and analyzed at soil lab (Faculty of Agriculture) in Al-Balqa Applied University. Depending on soil texture and grain size; the soil type of each site was determined according to USDA soil classification system (http://www.nrcs.usda.gov/wps/portal/nrcs/site/soils/home/), field capacity (FC) and the permanent wilt point (PWP) were also determined depending on soil type according to Israelsen (1932).

2.2.2.1.3 TYLCV transfection of tomato plants Seeds of tomato lines (GF13X 981 (T3), GF13 X981 (F7), and 981) were germinated in insect proof greenhouse. Three weeks old seedlings of each line were divided into two groups. Seedlings in group one were inoculated with TYLCV using viruliferous whiteflies (B. tabaci). The whiteflies were fed on TYLCV infected tomato plants that showed TYLCV symptoms after inoculating them with TYLCV infectious clones that have been provided from

Materials and methods

Dr. Fouad Akkad ( Hebrew University of Jerusalem) to the laboratory of Professor Ghandi Anfoka (Anfoka et al., 2009). Two days post inoculation; plants were sprayed with the insecticide Confidor (Bayer CropScience, Germany) to get rid of the whiteflies. Plants in the second group were not inoculated with TYLCV. All plants were kept in the green house until they were transferred to the field.

2.2.2.1.4 Plants in the field Plants were transplanted into the fields two weeks post inoculation. Plants in each field were divided into two parts: drought and watered. In each drought and watered treatment; plants were distributed randomly; each treatment was repeated three times. Each replicate at University site has 8 plants, while the replicates at Homra site have 7 plants in the drought part and 6 plants in the watered part.

2.2.2.1.5 Temperature monitoring Continuous measurements of the temperature were recorded daily during the experiment using DICKSON device (Addison, USA). Dickson device is a high-resolution temperature and humidity chart recorder, the recorder takes temperature readings every 50 seconds. An average of the readings taken between pen movements is what is drawn on the chart (http://www.dicksondata.com/products/TH8P5). The recorded data were transferred manually to excel file and presented in charts.

2.2.2.1.6 Monitoring the water content of the soil The water content of the soil was monitored during the period of the experiment using a Moisture Probe Meter (ICT International, Australia). Plants in drought treatment were subjected to 50% drought treatment. This was achieved depending on soil type. Every soil type has its own field capacity (FC) and the permanent wilt point (PWP). So the defining line between drought and watered treatment was calculated as the half way between FC and the PWP for each soil type. The soil water content of the plants in the drought parts was kept under the value of FC/2 but not under the PWP, while the soil water content of the plants in the watered area was kept over the value of FC/2. This soil water content was controlled by controlling the number and length of irrigation periods.

Materials and methods

2.2.2.1.7 Sample collection Leaf samples were collected from tomato plants one month after the transfer of the plants to the fields for transcriptome analysis as represented in figure 8 below. Six replicates of five discs (3mm in diameter) from five plants for each treatment were collected from each site. Five discs of each replicate were kept in RNA-Later solution (Qiagen) and shipped to Germany for microarray hybridization.

University Homra

Watered Drought Watered Drought

Infected Healthy Infected Healthy Infected Healthy Healthy Infected Figure 8: Outline of sample collection. For each tomato line, leaf samples were collected from the two selected sites (University, Homra) in both treatments (drought vs watered, healthy vs infected).

2.2.2.1.8 Symptoms monitoring

Plants were visually monitored for symptoms development 45 days after plants translocation in the fields. Disease severity index (DSI) was used to evaluate symptoms development on plants as described in Lapidot and Friedmann (2002). DSI= 0: no visible symptoms, DSI=1: very slight yellowing of leaflet margin or apical top, DSI=2: some yellowing and minor curling of leaflet ends, DSI=3: leaf yellowing, curling, and cupping with reduction in size, DSI = 4: severe stunting and yellowing of the plant, and pronounced cupping and curling.

2.2.2.2 Transcriptome analysis

2.2.2.2.1 RNA isolation Total RNA was isolated as described by Logemann et al., 1987 and then purified using RNAeasy Mini Spin Columns (Qiagen). RNA quantity was measured with the ND-100 spectrophotometer v3.3.0 (NanoDrop Technologies). RNA integrity was verified using an Agilent RNA 6000 Nano Chip on an Agilent 2100 BioAnalyzer (version B.02.03 BSI307) as recommended by the manufacturer’s protocol (Agilent RNA 6000 Nano Assay Protocol2).

Materials and methods

2.2.2.2.2 Microarray hybridization The cDNA, subsequent cRNA synthesis, and labeling were performed using the One- Color RNA Spike-Mix kit (Agilent) according to manufacturer's instructions. The Cy3-labeled cRNA was fragmented and hybridized to microarrays 4X44K tomato slides (Agilent) (17h at 65° C) and then washed according to the manufacturer's instructions. Slides were scanned on the Agilent Microarray Scanner with extended dynamic range at 5µm resolution. Data sets were extracted by the feature extraction software package (version 11.5.1.1 Agilent Technologies).

2.2.2.2.3 Analysis of Microarray data Scanned files were imported and analyzed using Genespring GX (version 12.6) software. EST sequences of differentially regulated genes were aligned to tomato genome in Sol Genomics database (http://solgenomics.net). Nucleotide sequences of the candidate genes that have been obtained from Sol Genomics were used to design oligonucleotide primers to study these genes.

2.2.2.2.4 DNA extraction, PCR amplification, and sequencing analysis Total DNA has been extracted from five tomato lines (GF13X981 (F7), 981, GF13X967, 967, and Favi-9) as described by Dellaporta et al., 1983. The extracted DNA was used as a template to amplify parts of the differentially expressed genes which are located on chromosome 11 (LRR (leucine rich repeat ) receptor-like serine/Threonine-protein Kinase, RLP, Splicing factor 3B subunit 4, RLK, Receptor like protein, Erwinia induced protein 2). TAKARA EX Taq polymerase (TAKARA BIO INC, Japan) was used to amplify the fragments according to manufacturer’s instructions and the following 30 X thermal cycles: 95o C for 1 min, 56o C for 45 sec, 72o C for 1 min with an initial step of 95o C for 5 min and final extension step of 72o C for 10 min. Biometra thermocycler (Göttingen, Germany) was used for all PCR reactions in this study. Amplified PCR fragments were extracted from gel and cleaned. PCR products (Erwinia induced protein 2 and receptor-like kinase) were sent for sequencing while the amplified fragments of LRR-RLP and Splicing factor 3B subunit 4 were ligated into pGEM-T Easy vector as recommended by the manufacturer. The recombinant plasmid preparations having the amplified PCR product of the gene of interest were sent for sequencing. Sequenced fragment were then analyzed using Geneious ® pro software (version 5.6.4).

Materials and methods

2.2.2.2.5 Prediction of the secondary structure of the LRR-RLP Expected translated polypeptide sequences resulted when the coding sequences of the TYLCV susceptible and resistant lines were analyzed to predict the secondary structure of the LRR-RLP using SOPMA in ExPASy website (http://www.expasy.org/proteomics).

2.2.2.2.6 Quantitative real-time RT-PCR (Q-RT-PCR) of the gene receptor-like kinase (RLK) Expression level of RLK was determined by Q-RT-PCR. Three biological replicates were used for the quantification of the transcript. Online available Primer3 Plus software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/) was used to design the primer for Q-RT-PCR. The oligonucleotides CaEF1a-2_5’ and CaEF1a-2_3’ (provided by Dr. Sophia Sonnewald, Data not published) were used to amplify elongation factor 1-alpha gene which used as referece to evaluate the expression level of RLK. The transcript was quantified by amplifying three technical replicates of cDNA of three biological replicates using gene specific primer in Brilliant II SYBR Green QPCR Master Mix (Agilent Technologies, USA).The thermal profile was as the following: 10 min at 95°C followed by 40 cycles of 15 sec. 95°C, 30 sec. 60°C and 15 sec. 72°C. The reaction has been made using BIORAD thermocycler. Relative gene expression was calculated using ΔCTmethod using reference gene as described in BIORAD Real-Time PCR Application Guide. Data of the quantitative real-time PCR were statistically analyzed using Microsoft excel t-test function, Significance levels P≤0.05was defined.

2.2.2.2.7 Analyzing the conserved domain of RLK and LRR-RLP

The protein sequences of RLK and LRR-RLP were analyzed in Conserved Domain search (CD-search) of NCBI (http://www.ncbi.nlm.nih.gov/cdd) to look for the functional domains of each protein. Protein sequences submitted in CD-search were obtained from sol genomics database for RLK (Solyc11g011880.1) and LRR-RLP (Solyc11g016930.1).

2.2.3 Transformation of Nicotiana tabaccum and Solanum lycopersicum with constructs having single or double genes

2.2.3.1 Preparation of the construct with the single gene (NSm) The double gene ΔCPIP1-HL-NSm construct was used as a template to amplify the movement protein of TSWV (NSm). After sequence verification, the fragment was ligated to pRB-35S vector. N. benthamiana plant has been agro infiltrated with Agrobacterium harboring the pRB-35S-NSm construct. Leaf disc samples were collected from infiltrated

Materials and methods plants daily for seven days after infiltration. Transient expression of NSm was detected by Western blot. For protein detection by Western Blot, total protein was extracted from leaf disc (5 mm) homogenized in 70 µl Lämmli buffer (200mM Tris-HCl, pH=6.8; 18% ß- Mercaptoethanol (v/v); 40% Glycerin (v/v); 0.01% Bromphenol blue (w/v); 8% SDS (w/v)). Samples were separated on 10 % (v/v) SDS-PAGE and blotted onto nitrocellulose membranes (Macherey-Nagel, Düren, Germany). Membranes were blocked overnight at 4°C in 5% skimmed milk/TBST (20 mM Tris, pH 7; 500 mM NaCl; and 0.1% Tween 20), and incubated for 60 minutes at room temperature in Anti-HA antibodies (Roche) diluted 1:500 in 1% skimmed milk/TBST. ECL immunodetection was performed according to standard procedures.

2.2.3.2 Stable transformation of N. tabaccum and S. lycopersicum plants

2.2.3.2.1 Stable transformation of tobacco and tomato plants Agrobacterium-mediated transformation method was used to transform tobacco plants with the constructs pRB35S:ΔCPIP1-NSm, pRB35S:ΔM541 and pRB35S: NSm, and to transform tomato plants with the constructs pRB35S:ΔCPIP1-NSm, pRB35S:ΔM541 and pRB35S: ΔCPIP.

2.2.3.2.2 Analyzing regenerated plantlets Following transformation, regenerated plantlets were tested whether they are transformed or not. Tobacco plant that have been transformed with the constructs pRB35S:ΔCPIP1-NSm, pRB35S:ΔM541 and pRB35S: NSm and tomato plants that have been transformed with the constructs pRB35S:ΔCPIP1-NSm, and pRB35S:ΔM541were tested for the expression of the target protein via western blot (as mentioned above in part 2.2.3.1), while tomato plants that have been transformed with the construct pRB35S: ΔCPIP were detected for the transcript via reverse transcriptase PCR using gene specific primer. For transcript detection, total RNA was isolated from ΔCPIP1 regenerated tomato plantlets as described by Logemann et al. (1987). Total RNA was subjected to DNAseI (Fermentas) treatment following manufacturer’s instructions. DNAseI-treated RNA was transcribed into cDNA using RevertAid ™ H Minus M-MuLV Reverse Transcriptase (Fermentas) according to manufacturer’s protocols. Random hexamer primer (Fermentas) was used for cDNA synthesis. Gene specific oligonucleotides for ΔCPIP1 have been used to amplify ΔCPIP1 using following thermal cycles 10 min at 95°C followed by 30 cycles of

Materials and methods

95°C for 45 sec., 56°C for 45 sec. and 72°C for 45 sec. with final extension 72°C for 10 min. plantlets that showed protein expression or the presence of the transcript were transferred to the greenhouse for seed collection. Plantlets that expressed the target protein were duplicated. Duplicated plantlets were transferred to the greenhouse to be tested for virus resistance and for seeds collection.

2.2.3.2.3 Testing transgenic N. tabaccum plants for TSWV resistance Transgenic N. tabaccum plants (expressing ΔCPIP1-NSm, ΔCPIP1-ΔM541, ΔM541 and NSm) were tested for TSWV resistance. Transgenic plants were mechanically inoculated with TSWV inoculum that has been provided by DSZM Company (Braunschweig, Germany). The mechanical inoculation has been performed as recommended by the supplier. Briefly, lyophilized leaves of TSWV inoculum were homogenized in inoculation buffer; Carborundum is added to the homogenate and then rubbed on fully developed leaves of tobacco plants. Inoculated leaves were rinsed with water and then monitored for symptom development. Regenerated transgenic tobacco plantlets that expressed (proofed by Western Blot) the double gene construct ΔCPIP1-NSm and the single gene constructs ΔM541, and NSm were used to be tested for TSWV resistance. T1 generation tobacco plants expressing the double gene construct ΔCPIP1-ΔM541 were used to proof the viral resistance of TSWV. Four lines of ∆ CPIP1-∆M541 T1 transgenic tobacco plants were grown in the greenhouse and mechanically inoculated with TSWV, observed for symptom and then the expression of the protein ∆ CPIP1- ∆M541 was detected by western blot. Nine plants from each ∆ CPIP1- ∆M541 T1 transgenic tobacco lines were used for testing TSWV resistance, 7 plants were mechanically inoculated with TSWV inoculum, one plant was mock inoculated with the inoculation buffer, and one plant has been not inoculated.

2.2.3.3 Selection of homozygous transgenic lines Transgenic tobacco plants expressing ΔCPIP1-NSm and NSm were selected for homozygosity. Three transgenic lines have been used for this purpose. Seeds collected from regenerated plants were germinated and grown in the green house, detected for protein expression by western blot (as mentioned above in part 2.2.3.1). Positive T1 plants were grown in the greenhouse and T2 seeds were collected. Transgenic tomato plants expressing the resistance genes (ΔCPIP1) were selected for homozygosity. Three transgenic lines have been used for this purpose. Seeds collected from

Materials and methods regenerated plants were germinated on MS media containing 50mg/L Kanamycin; germinated plantlets were grown in the greenhouse to collect T2 seeds.

2.2.4 Identification of host factors interacting with TYLCV

2.2.4.1 DNA Isolation Leaves of TYLCV infected tomato (collected from Jordan field experiment) were used to isolate DNA according to Dellaporta et al., (1983).

2.2.4.2 Rolling circle amplification (RCA) of TYLCV DNA Total DNA extract that has been prepared from tomato leaves was subjected to rolling circle amplification reaction using illustra TempliPhi 100 Amplification Kit (GE Healthcare Life Sciences, UK) according to manufacturer’s instructions. Healthy tomato plants were used as a negative control and the vector pUC19 (a component of the illustra TempliPhi 100 Amplification Kit) was used as a positive control for this reaction).

2.2.4.3 Sequencing of TYLCV isolates RCA product was digested by SphI (Fermentas, Germany) following manufacturer’s instructions. SphI digested RCA products of TYLCV DNA were ligated to the vector pUC19 using Quick ligation kit (NEB, UK) as recommended by the manufacturer. The ligation reaction was used to transform E.coli. Plasmids prepared using alkaline lysis method (Birnboim and Doly, 1979) from overnight broth cultures of white colonies grown on LB/Ampicillin plate containing IPTG and X-gal. The restriction endonuclease EcoRI (Fermentas, Germany) was used to check positive clones according to manufacturer’s instructions.The prepared plasmid was sent for sequencing. M13 forward and reverse primers have been used to get the first sequence which was used later on to design the oligonucleotides (TY-FWP1, TY-RWP1, TY-FWP2, TY- RWP2, TY-FWP3, TY-RWP3) to have the complete sequence of TYLCV isolate. Each sequencing reaction has been repeated three times to proof the sequence.

2.2.4.4 Cloning the open reading frames (ORFs) of TYLCV Sequenced TYLCV genome was used to design the oligonucleotides used to amplify the ORFs (V1, V2, C2) of TYLCV using pfu DNA polymerase (Promega) as described by the manufacturer. Thermal cycles were 30 as follows: 95o C for 1 min, 56o C for 1 min, 72o C for 1 min with final extension of 10 min at 72o C. The amplified fragments of the ORFs V1, V2, and C2 were cloned into pENTR-D-TOPO using pENTR™ Directional TOPO® Cloning Kits,

Materials and methods and then cloned into the destination vectors pK7FWG2 (V1), pK7WGF2 (V2, C2) pGWB661 (V2, C2) using Gateway® LR Clonase® II Enzyme mix (Invitrogen). The vectors (V1-eGFP: pK7FWG2, eGFP-V2:pK7WGF2, eGFP-C2: pK7WGF2, TagRFP-C2: pGWB661 and TagRFP-V2: pGWB661) have been used to transform Agrobacterium tumefaciens.

2.2.4.5 Subcellular Localization of TYLCV ORFs Plants of N. benthamiana were agro-infiltrated with Agrobacteria harboring the constructs (V1-eGFP: pK7FWG2, eGFP-V2:pK7WGF2, eGFP-C2:pK7WGF2, TagRFP-C2: pGWB661 and TagRFP-V2: pGWB661) individually. Infiltrated leaves were sampled 48 hours after agro-infiltration, examined under the confocal laser scanning microscopy (CLSM) for sub-cellular localization of the fused proteins. CLSM work has been made in optical imaging center Erlangen (OICE).

2.2.4.6 Immunoprecipitation of fused protein of V1, V2, and C2 Three biological replicates from each infiltrated leaves harboring V1-GFP, GFP-V2, GFP-C2 have been immune-precipitated using anti- GFP coupled to magnetic micro particles using GFP-Trap®_M (Chromotek, Germany). For immune-precipitation, infiltrated leaves (100mg) were homogenized in 200 µl lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) mixed with 1 mg/ml DNase (Fermentas, , Germany), 2.5 mM MgCl2, protease inhibitor cocktail tablets (one tablet for each 10 ml lysis buffer) (Roche, Germany). The homogenized mix was placed at 4°C for 30 min with extensive mixing every 10 minutes; the volume of lysate supernatant was adjusted to 500 µl with dilution buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA). The diluted lysate supernatant was mixed with pre-equilibrated GFP-Trap M beads. The equilibration was carried out by re-suspending 25µl of bead mixture in 500 µl ice cold dilution buffer. Beads were then separated on magnetic rack and washed for 3 times with 500 µl ice cold dilution buffer. The mixture of diluted lysate supernatant with equilibrated beads was incubated for one hour at 4°C with constant mixing. The mixture was centrifuged t 2.500 g for 5 min at 4°C; beads were washed three times with 500 µl ice cold washing buffer (same composition of dilution buffer). The bound proteins were eluted by adding 50 µl 0.2 M glycine pH 2.5 (incubation time: 30 sec under constant mixing) followed by centrifugation. The eluent was neutralized by 5 µl 1M Tris-base pH 10.4. The elution step was repeated twice.

Materials and methods

RFP-C2 and RFP-V2 were immune-precipitated from corresponding agro-infiltrated leaves of N. benthamiana using anti- RFP coupled to magnetic micro particles using RFP- Trap®_M (Chromotek, Germany).

2.2.4.7 Tryptic digestion of the precipitated protein Pierce trypsin protease, MS grade (Fermentas, Germany) was used to digest the precipitated protein preparations. Trypsin (20µg) was reconstituted in 1ml digestion buffer (830 µl water, 100µl Acetonitrile, 70µl of 400mM NH4NCO3), and then 10µl was added for each sample. Samples were incubated over night at 37OC, dried in speed vacuum for 4 hours and then resuspended in 50µl formic acid (5%).

2.2.4.8 Analysis of precipitated protein via Nano LC-Mass Spectrometry Trypsin digested protein preparations (10µl) were injected into a Dionex Ultimate 3000 nanoHPLC System connected to an Orbitrap FusionTM TribridTM mass spectrometer with nanospray FlexTM ion source (Thermo Fisher Scientific, Bremen, Germany). Controlling software was Chromeleon VS6.8, DCMS-Link VS1.1, and Xcalibur VS1.0. Peptides were desalted online by an Acclaim PepMap100 C18 trap column (300µm i.d. x 5mm, 5 µm particles, and 100 Å pores) with 0.1 % trifluoracetic acid at a flow rate of 20µl/min generated by the loading pump. After 5 minutes the trap was switched into the stream of the nano pump and then separated at 35ºC on an Acclaim PepMap RSLC C18 column (75µm i.d. x 15cm, 2µm, 100 Å, Thermo Fisher) with a gradient formed by 0.1% formic acid (eluent A) and acetonitril in 0.5% formic acid (eluent B) at 300nL/min. Eluent B was 3% for 5min, 35% from 5-125min, 80% from 125-130min, 80% from 130-140min, 3% from 140-141min and 3% from 141-160min. Gas phase cations of peptides were formed at a voltage of 2.0k in the ion source and sprayed into the mass spectrometer with a transfer capillary temperature of 275ºC.The instrument was operated in a data-dependent mode in top speed performing full scans and dependent MS2 scans in cycles limited to 3 seconds. Full scans for precursor ions from 300- 2000 m/z were performed at a resolution of 120K (at 200m/z) and a target value (AGC) of 4x105 ions with a maximum fill time of 50ms for the Orbitrap detector. Monoisotopic precursors with an intensity of more than 5000 and the charge states from 2 to 7 were selected by the quadrupole with a mass tolerance of 10 ppm, an isolation window of 1.6 m/z and a dynamic exclusion for 120s. For the dependent MS2 scans, they were activated by HCD (higher-energy collisional dissociation) with normalized collision energy of 27% in the ion- routing multipole. With a maximum fill time of 250ms and AGC (automatic gain control)

Materials and methods target value of 1.0e4 for the Ion-trap product ions finally were resolved after accumulation in the ion trap detector with a scan rate set to rapid.Calibration of the instrument was achieved with an internal lock-mass ion of 445.12003 (m/z) together with external calibration according to the manufacturer’s instructions.

2.2.4.9 Data analysis The raw data for MS2 spectra were searched against N. benthamiana database release containing 56708 entries of protein sequences, downloaded November 2014 including the added controls using Proteome Discoverer (version 1.4.14. Thermo Fisher Scientific) and its SEQUEST search engine. Theoretical masses for peptides generated by Trypsin with a maximum of 2 missed cleavage sites and their product ions were compared to the measured spectra with the following parameters: Static modification with carbamidomethylation at cysteine (mass shift + 57.021 Da). Mass tolerance was set to 10 ppm for precursors and 0.6 Da for product ions. For increased confidence validation of peptide spectral matches (PSMs) by the percolator algorithm, applying the target-decoy search strategy was based on q-values at a target FDR of 1%. Identified peptides were finally grouped into proteins applying the law of parsimony (2) and filtered to 1% FDR. Only proteins with at least 1 identified peptide with at least 6 amino acids were considered.

Results

3. Results

3.1 Transcriptome analysis of tomato plants exposed to triple stress A field experiment had been carried out in Jordan during the period May to August in the year 2012 to study the impact of different stress conditions on the transcriptome profile of tomato plants. Tomato plants were exposed to TYLCV, heat and drought stress. Ninety-six seedlings (3-weeks old) of two resistant tomato lines (GF13X981-F7, GF13X981-T3) and a susceptible tomato line (981) were inoculated with TYLCV using viruliferous whiteflies in the greenhouse of the Research Station of the Faculty of Agricultural Technology in Al- Al- Balqa’ Applied University (Al-Salt City, Jordan). An equal number of seedlings were mock- inoculated. One month later, plants were transferred to the field in two selected locations. The first site was the Campus of Al-Balqa’ Applied University (900 m above sea level), while the other site was the Research Station of the Faculty of Agricultural Technology in Al- Al-Balqa’ Applied University (Homra), 200 m above Sea level. In both sites, plants were divided into two groups and treated differently: drought and watered. Plants were distributed in the field randomly (randomized block design), each treatment has three replicates in both sites. The distribution of tomato plants in the field was as presented in figure 9. Field capacity (FC) and the permanent wilting point (PWP) of the soil at both sites were already determined before transferring tomato plants from the green house to the fields. Soil samples were collected and analyzed in the Soil laboratory of the Faculty of Agricultural Technology (Al- Balqa’ Applied University). According to the USDA classification system, which is based on grain size, the soil type at the University site was classified as clay soil, while that of Homra site was classified as loamy soil. For the University site, the FC and PWP were 35.1% and 17.2%, respectively, while that for Homra site were 22.3% and 10.2%. Based on soil type, field capacity and permanent wilting point, plants under drought conditions were subjected to 50% drought. 50% drought treatment has been calculated as the half way between field capacity and permanent wilting point for each soil type. For example, at University site, soil water content was kept between 17.2-26% and 26%-35.1% in the groups under drought and watered conditions, respectively. During the experiment, the soil water content was monitored daily using a Moisture Probe Meter to control water application according to the moisture content's daily readings.

Results

GF13 X 981 T3 GF13 X 981 F7 981 GF13 X 981 T3 GF13 X 981 F7 981

(I) (H) (I) (H) (I) (H) GF13 X 981 F7 981 GF13 X 981 T3 GF13 X 981 F7 981 GF13 X 981 T3 (H) (I) (H) (I) (H) (I)

Drought 981 GF13 X 981 T3 GF13 X 981 F7 981 GF13 X 981 T3 GF13 X 981 F7 (I) (H) (I) (H) (I) (H) University field (8 plants/ replicate)

981 GF13 X 981 F7 GF13 X 981 T3 981 GF13 X 981 F7 GF13 X 981 T3 (H) (I) (H) (I) (H) (I) GF13 X 981 T3 981 GF13 X 981 F7 GF13 X 981 T3 981 GF13 X 981 F7 (I) (H) (I) (H) (I) (H) GF13 X 981 F7 GF13 X 981 T3 981 GF13 X 981 F7 GF13 X 981 T3 981 Watered (H) (I) (H) (I) (H) (I)

Drought (7 plants / replicate) Watered (6 plants/replicate)

GF13X981 GF13X98 981 GF13X981T GF13X981 GF13X981 981 GF13X981 T3 F7 (I) 3 T3 F7 (I) T3 (I) (H) (H) (I) (H) (H)

981 981 GF13X981 GF13X981 981 981 GF13X981 GF13X981 (H) (H) F7 T3 (H) (H) F7 T3 (I) (H) (I) (H) GF13X981 981 GF13 X981 GF13X981 GF13X981 981 GF13X981 GF13X981 F7 (H) T3 F7 F7 (H) T3 F7 (I) (I) (H) (I) (I) (H) Homra field field Homra 981 GF13X981 981 GF13X981 981 GF13X981 981 GF13X981 (I) T3 (H) F7 (I) T3 (H) F7 (H) (I) (H) (I) 981 GF13X981 GF13X981 981 981 GF13X981 GF13X981 981 (H) F7 T3 (I) (H) F7 T3 (I) (H) (I) (H) (I) Figure 9: Scheme representation of the distribution of the plants from different treatments at the university field (upper panel) and at Homra field (lower panel). Bold written 981 (H) in Homra field were planted just to fill the gap and were not used in any analysis. H and I referred to healthy and infected plants respectively. 3.1.1 Temperature records at both sites The temperature was continuously measured and recorded to evaluate the heat stress that tomato plants were exposed to at each site. Temperature records showed that the average of the highest temperature at University site (assumed to be the controlled non-stressed temperature site) during the day was 41.5 °C, while the average highest temperature during the night was 20.6 °C. At Homra site, the average highest temperature during the day was 44.8 °C, and the average during the night was 25.2 °C. Figures 10 and 11 showed the recorded temperatures in both sites day by day during the experiment.

Results

60 50

20 Night Temp (°C) Temp 10 Day 0

Date

Figure 10: Temperature records at the University site during the experiment.

60 50

40 30 20 (°C) Temp Night 10 Day

Date

Figure 11: Temperature records at Homra site during the experiment.

3.1.2 Evaluation of disease symptoms Tomato plants were monitored for symptoms development of tomato yellow leaf curl virus disease (TYLCD) 45 days after transferring plants to the fields. Disease severity index (DSI) was used to rate the plant for symptoms as described by Lapidot and Friedmann (2002). DSI scale is shown in figure 12. The evaluations of disease severity at the University and Homra sites (tables 6 and 7) showed that plants of the susceptible lines (981) in both sites have the DSI value 3 and 4. This means that plants from the susceptible line (981) were showing severe TYLCV symptoms although they were not inoculated with TYLCV, TYLCV infection of non-inoculated plants has been proofed by PCR in the final phases of the experiment. For the resistant lines (GF13 X981 (T3) and GF13 X 981 (F7)) at both sites and in each treatment, few plants showed DSI value 0. The maximum number of resistant plants that have DSI =0 is eight plants of T3 resistant line in the watered part at university site. Plants from the resistant

Results lines (T3 and F7) were showing a variable degree of disease severity even for plants that were non-inoculated. Ten watered F7 plants (resistant line) at Homra field were showing DSI=4.Tables 1 and 2 indicate that a large number of plants from the susceptible line 981 ( up to 18plants) did not even survive during the time of the experiment, whereas the number of dead plants from the resistant lines ranged between 0 to 6 plants).

0 1

2 3 4

Figure 12: Scale of disease severity index (DSI) for symptoms evaluation of TYLCD in tomato plants. DSI= 0: no visible symptoms, DSI=1: very slight yellowing of leaflet margin or apical top, DSI=2: some yellowing and minor curling of leaflet ends, DSI=3: leaf yellowing, curling, and cupping with reduction in size, DSI = 4: severe stunting and yellowing of the plant, and pronounced cupping and curling.

Results

Treatment Tomato line Line DSI (0-4) No. of type 0 1 2 3 4 dead R/S plants GF13 X 981 (T3) (I) R 4 11 5 4 0 0 GF13 X 981 (T3) (H) R 1 13 9 1 0 0

GF13 X 981 (F7) (I) R 4 13 2 5 0 0 Drought GF13 X 981 (F7) (H) R 0 5 9 9 0 1 981 (I) S 0 0 0 0 14 10 981 (H) S 0 0 0 17 7 0

University University GF13 X 981 (T3) (I) R 8 10 2 3 1 0 GF13 X 981 (T3) (H) R 0 8 7 8 0 1 GF13 X 981 (F7) (I) R 4 15 4 1 0 0 Watered GF13 X 981 (F7) (H) R 1 9 4 10 0 0 981 (I) S 0 0 0 0 21 3 981 (H) S 0 0 0 22 2 0 Table 6: Evaluation of TYLCV symptoms at University site. Numbers refer to the number of plants having this value of disease severity index. I: TYLCV infected plants, H: healthy plants. Line type refers to resistant (R) or susceptible (S) lines.

Treatment Tomato line Line DSI (0-4) No. of type 0 1 2 3 4 dead R/S plants GF13 X 981 (T3) (I) R 1 4 2 8 1 5 GF13 X 981 (T3) (H) R 1 5 6 6 1 2 GF13 X 981 (F7) (I) R 3 8 1 3 0 6 Drought GF13 X 981 (F7) (H) R 1 4 4 5 4 3 981 (I) S 0 0 0 3 0 18 981 (H) S 0 0 0 1 17 3 Homra Homra GF13 X 981 (T3) (I) R 0 2 5 9 0 2 GF13 X 981 (T3) (H) R 0 0 0 7 8 3 GF13 X 981 (F7) (I) R 0 3 6 4 0 5 Watered GF13 X 981 (F7) (H) R 0 0 2 6 10 0 981 (I) S 0 0 0 0 5 13 981 (H) S 0 0 0 0 16 2 Table 7: Evaluation of TYLCV symptoms at Homra site. Numbers refer to the number of plants having this value of disease severity index. I: TYLCV infected plants, H: healthy plants. Line type refers to resistant (R) or susceptible (S) lines. 3.1.3 Transcriptome analysis of field samples To understand the molecular response of tomato plants exposed to triple stress conditions (virus, heat, and drought), microarray hybridizations were performed using RNA samples isolated from leaf samples collected from plants grown in seventeen environmental conditions. Samples collected from University site were assumed to be the samples of the low

Results heat stress when compared to samples collected from Homra. Plant samples representing environmental conditions that have been analyzed are listed in table 8. Conditions No. Site Tomato line Watering TYLCV Abbreviation 1 University 981 Drought Healthy U 981 D H 2 University 981 Drought Infected U 981 D I 3 University GF13X981 F7 Drought Healthy U F7 D H 4 University GF13X981 F7 Drought Infected U F7 D I 5 University GF13X981 T3 Drought Healthy U T3 D H 6 University GF13X981 T3 Drought Infected U T3 D I 7 University GF13X981 T3 Watered Healthy U T3 W H 8 University GF13X981 T3 Watered Infected U T3 W I 9 Homra 981 Drought Healthy H 981 D H 10 Homra 981 Drought Infected H 981 D I 11 Homra 981 Watered Healthy H 981 W H 12 Homra 981 Watered Infected H 981 W I 13 Homra GF13X981 T3 Drought Infected H T3 D I 14 Homra GF13X981 T3 Watered Healthy H T3 W H 15 Homra GF13X981 T3 Watered Infected H T3 W I 16 Homra GF13X981 F7 Watered Healthy H F7 W H 17 Homra GF13X981 F7 Watered Infected H F7 W I Table 8: Environmental conditions that have been analyzed for transcriptional changes. Abbreviation is a short name for each treatment summarizes the condition that the samples refer to using the underlined initial of each condition.U: university site, First H: Homra site, T3 and F7: TYLCV resistant lines, 981: TYLCV susceptible line, D: drought, W: watered, second H: healthy or non TYLCV inoculated, I: TYLCV infected.

Data were analyzed using GeneSpring software (GX 12.6). A condition tree based on hierarchical clustering analysis of expression values of all entities (Figure 13) shows that sample grouping is not influenced by the environmental conditions. Clustering pattern indicates that differently treated plants grouped together in the same clusters. Plants that have been subjected to high heat stress (Homra samples) were found to be grouped in the same cluster with those grown in the lower heat stress field (University samples). It is also shown that well-watered plants were grouped with plants that were in the drought part of the fields.

Results

Figure 13: Condition tree based on hierarchical clustering of expression data of samples from different environmental conditions. Samples were clustered on conditions, Distance matric is Pearson’s centered Absolute, with Centroid linkage rule. U: university site, First H: Homra site, T3 and F7: TYLCV resistant lines, 981: TYLCV susceptible line, D: drought, W: watered, second H: healthy or non TYLCV inoculated, I: TYLCV infected.

Results

3.1.4 Differential gene regulation in resistant and susceptible lines As shown in the hierarchical condition tree of the samples (Figure 13), Samples representing the same treatment are not grouped in one cluster. These samples were combined together as one replicate for each environmental condition as shown in table 8 above and a differential entity list has been extracted. From this differential list, thirteen entities were shown to be differentially regulated in the resistant lines T3 and F7 when they were compared to the susceptible line (981) regardless of the conditions that plants were grown in (heat, drought) and whether these plants were TYLCV infected or healthy. The differentially regulated entities are down regulated in susceptible lines while they are up regulated in resistant line The EST sequences of the differentially regulated entities obtained from tomato annotation files were aligned against the sol genomics database to identify the genes corresponding to these entities. The differential expression of these genes is presented in the linear graph (Figure 14) and heat map (Figure15). Figures 14 and 15 indicate that these genes are down regulated in the susceptible line (981) compared to their expression level in the resistant lines (T3 and F7). Samples that showed down regulation of the differentially regulated genes are numbered 1-6 in figure 14 referred to the resistant line 981 and they were grown in variable stress conditions. Samples that showed up regulation of differentially regulated genes are numbered 7-17 in figure 14 referred to the resistant lines T3 and F7 and have been grown under different stress conditions also. As shown in figure 15, the transcript level of differentially regulated genes (listed on the left panel) in susceptible lines is presented in blue color for down-regulation, while the transcript level of same genes which are up-regulated in resistant lines is represented in red color. Table 9 below lists these genes and describes on which chromosome these genes are located, in addition to the locus of each gene on the chromosomes of tomato genome.

Results

Figure 14: linear graph representing the expression levels of differentially regulated genes in susceptible and resistant lines. Samples 1-6: susceptible 981 plants, 7-17: Resistant (GF13 X981 (T3) or GF13 X981 (F7)) plants.

Figure 15: Heat map representation of expression level of genes that are differentially regulated in susceptible line (981) compared to resistant lines (T3, F7). The color scale at the top of the panel indicates transcript levels; red representing an increase and blue representing a decrease in transcript levels. The colors saturate at 2-fold change. Transcript level of differently treated 981 susceptible lines is down-regulated (blue), and that for differently treated resistant lines (T3, F7) is up-regulated (red).

Results

No. Chromosome Locus on the Gene No. Chromosome 1 18,523,461- unknown protein Chr.1 18,523,999 2 27,559,591- unknown protein Chr.1 27,562,975 3 tRNA pseudouridine synthase B; contains Interpro 40,195,042- Chr.2 domain(s) IPR004802 Pseudouridine synthase, putative 40,195,773 4 4,981,234- Rho-related GTP-binding protein RhoC (AHRD V1 **-- 4,985,169 RHOC_PONAB); contains Interpro domain(s) Chr.3 IPR003578 Ras small GTPase, Rho type

5 Glycosyl hydrolase family 5 protein/ Cellulase family 7,117,665- Chr.5 protein 7,117-995 6 26,351,466- PH domain containing protein Chr.5 26,366,692 7 3,944,263- unknown protein Chr.11 3,944,990 8 4,040,668- Erwinia induced protein 2 Chr.11 4,041,263 9 RLK, Receptor like protein, putative resistance protein 4,818939- Chr.11 with an anti-fungal activity 4,822147 10 LRR (Leucine rich repeat ) receptor-like 7,594,055- Chr.11 serine/Threonine-protein Kinase,RLP 7,599,337 11 Splicing factor 3B subunit 4 (AHRD V1 *--- 27,843,051- Q22R82_TETTH); contains Interpro Chr.11 27,845,108 domain(s) IPR000504 RNA recognition motif, RNP-1 12 34,093,539 - Unknown protein Chr.11 34,094,101 13 CRABS CLAW (Fragment) contains interpro domain(s) 55,165,970- Chr.11 IPR006780 YABBY protein 55,172,156 Table 9: Genes that are differentially regulated in resistant and susceptible tomato lines, their chromosome location and locus on the chromosome. 3.1.5 Differentially regulated genes located on chromosome 11 Table 9 reveals that seven out of thirteen differentially regulated genes are located particularly on tomato chromosome number 11. These seven genes are summarized in table 10 with their Sol Genomics identifiers. The exact locus of these genes (Table 9) indicates that the majority of differentially regulated genes are distributed along chromosome 11. The scattered distribution of those genes along chromosome 11 removes away the possibility that the resistance phenotype is inherited as one locus on this chromosome. So, the up regulated genes in resistant lines are not located within a specific marker that defines the resistance phenotype.

Results

# Gene Name Sol Genomics

1 unknown protein Solyc11g010870.1

2 Erwinia induced protein 2 Solyc11g011010.1 3 RLK, Receptor like protein, putative

Solyc11g011880.1 resistance protein with an anti-fungal activity 4 LRR receptor-like serine/Threonine-protein

Solyc11g016930.1 Kinase, RLP 5 Splicing factor 3B subunit 4, RNA

Solyc11g039950.1 recognition motif, RNP-1

6 Unknown protein Solyc11g042860.1 7 CRABS CLAW fragment contains interpro

Solyc11g071810.1 domain (YABBY protein) Table 10: differentially regulated genes that are located on chromosome 11 as they were identified in the sol genomics database. To examine if the up-regulated genes are introduced to the resistant breeding lines from the resistant parent, sequence identity analysis was performed for the candidate genes Erwinia induced protein 2 Solyc11g011010.1, RLK (Solyc11g011880.1), LRR-RLP (Solyc11g016930.1), and the Splicing factor (Solyc11g039950.1). Fragments of the genes were amplified from genomic DNA of the five tomato lines 981 and 976, which are TYLCV susceptible, the breeding lines GF13X981 F7 and GF13X967 F5, which are TYLCV resistant, and Favi-9, one of the resistant parental lines (plant materials were kindly provided by Prof. Ghandi Anfoka, Faculty of Agricultural technology, Al-Balqa’ Applied University). The analysis of nucleotide alignments of the sequenced fragments of the candidate genes (Erwinia induced protein 2, RLK, Splicing factor) showed that the sequences of the resistant lines (GF13 X 981 (F7), GF13 X 967 (F5), Favi-9) shared high sequence identity of more than 99% with those of the susceptible lines (981, 967). Nucleotide alignments of these sequences are shown in the appendix. Despite the high sequence similarity between the amplified fragments of LRR-RLP (Supplement 1) from the resistant lines (GF13 X 981 (F7), GF13 X 967 (F5), Favi-9) and the susceptible lines (981, 967), nine single nucleotide polymorphisms (SNPs) were detected in these fragments. These SNPs were conserved in the tested resistant lines (F5, F7, and Favi-9) in comparison to the susceptible lines (981, 967). In addition, the fragments size obtained from resistant and susceptible lines was different, 815 bp compared to 808 bp, respectively. This size difference was due to seven additional bases located in intron no. 2 of this gene.

Results

1 10 20 30 40 50 60 | | | | | | | 967 ATGTGGGCATGTATCAAGTTCTTTATCTCTCAAGAATTTCGAAGCAGAAGTTGTGAATGT 981 ATGTGGGCATGTATCAAGTTCTTTATCTCTCAAGAATTTCGAAGCAGAAGTTGTGAATGT F5 ATGTGGGCATGTATCAAGTTCTTTATCTCTCAAGAATTTCGAAGCAGAAGTTGTGAATGT F7 ATGTGGGCATGTATCAAGTTCTTTATCTCTCAAGAATTTCGAAGCAGAAGTTGTGAATGT Favi-9 ATGTGGGCATGTATCAAGTTCTTTATCTCTCAAGAATTTCGAAGCAGAAGTTGTGAATGT Solyc11g016930.1 ATGTGGGCATGTATCAAGTTCTTTATCTCTCAAGAATTTCGAAGCAGAAGTTGTGAATGT

967 CTTCTAGATTATTGTCAAAGGTTGCTACTTCACAATGTTACTGCATCAAATGATATTAAG 981 CTTCTAGATTATTGTCAAAGGTTGCTACTTCACAATGTTACTGCATCAAATGATATTAAG F5 CTTCTAGATTATTGTCAAAGGTTGCTACTTCACAATGTTACTGCATCAAATGATATTAAG F7 CTTCTAGATTATTGTCAAAGGTTGCTACTTCACAATGTTACTGCATCAAATGATATTAAG Favi-9 CTTCTAGATTATTGTCAAAGGTTGCTACTTCACAATGTTACTGCATCAAATGATATTAAG Solyc11g016930.1 CTTCTAGATTATTGTCAAAGGTTGCTACTTCACAATGTTACTGCATCAAATGATATTAAG

967 TGCTTGCAAGGACTGAAGGACTCATTTAAGGATCCTAATGTAAATTTCAATTCTTGGAAC 981 TGCTTGCAAGGACTGAAGGACTCATTTAAGGATCCTAATGTAAATTTCAATTCTTGGAAC F5 TGCTTGCAAGGACTGAAGGACTCATTTAAGGATCCTAATGTAAATTTCAATTCTTGGAAC F7 TGCTTGCAAGGACTGAAGGACTCATTTAAGGATCCTAATGTAAATTTCAATTCTTGGAAC Favi-9 TGCTTGCAAGGACTGAAGGACCCATTTAAGGATCCTAATGTAAATTTCAATTCTTGGAAC Solyc11g016930.1 TGCTTGCAAGGACTGAAGGACTCATTTAAGGATCCTAATGTAAATTTCAATTCTTGGAAC

967 TTCTCAAACTACTCCATGGGGTTTATCTGCAAGTTTGTTGGTGTCATTTACTGGAACAAC 981 TTCTCAAACTACTCCATGGGGTTTATCTGCAAGTTTGTTGGTGTCATTTACTGGAACAAC F5 TTCTCAAACTACTCCATGGGGTTTATCTGCAAGTTTGTTGGTGTCATTTACTGGAACAAC F7 TTCTCAAACTACTCCATGGGGTTTATCTGCAAGTTTGTTGGTGTCATTTACTGGAACAAC Favi-9 TTCTCAAACTACTCCATGGGGTTTATCTGCAAGTTTGTTGGTGTCATTTACTGGAACAAC Solyc11g016930.1 TTCTCAAACTACTCCATGGGGTTTATCTGCAAGTTTGTTGGTGTCATTTACTGGAACAAC

967 CTCGAGAACCGGATGATCAGCCTCTCACTTCCAAACATGAATCTCAGTGTACAGCTCCCA 981 CTCGAGAACCGGATGATCAGCCTCTCACTTCCAAACATGAATCTCAGTGTACAGCTCCCA F5 CTCGAGAACCGGATGATCAGCCTCTCACTTCCAAACATGAATCTCAGTGTACAGCTCCCA F7 CTCGAGAACCGGATGATCAGCCTCTCACTTCCAAACATGAATCTCAGTGTACAGCTCCCA Favi-9 CTCGAGAACCGGATGATCAGCCTCTCACTTCCAAACATGAATCTCAGTGTACAGCTCCCA Solyc11g016930.1 CTCGAGAACCGGATGATCAGCCTCTCACTTCCAAACATGAATCTCAGTGTACAGCTCCCA

967 GATGCCTTCAAATGTTGCTCATCTTTGGCTACTCTTCATCTCTCTGGTAACAGTTTCTCT 981 GATGCCTTCAAATGTTGCTCATCTTTGGCTACTCTTCATCTCTCTGGTAACAGTTTCTCT F5 GATGCCTTCAAATGTTGCTCATCTTTGGCTACTCTTCATCTCTCTGGTAACAGTTTCTCT F7 GATGCCTTCAAATGTTGCTCATCTTTGGCTACTCTTCATCTCTCTGGTAACAGTTTCTCT Favi-9 GATGCCTTCAAATGTTGCTCATCTTTGGCTACTCTTCATCTCTCTGGTAACAGTTTCTCT Solyc11g016930.1 GATGCCTTCAAATGTTGCTCATCTTTGGCTACTCTTCATCTCTCTGGTAACAGTTTCTCT

967 GGCTCTATACCTGCTGAACTTAGAAACTGCACCTACTTGAACAAGTTGATTCTCAATAAC 981 GGCTCTATACCTGCTGAACTTAGAAACTGCACCTACTTGAACAAGTTGATTCTCAATAAC F5 GGCTCTATACCTGCTGAACTTGGAAACTGCACCTACTTGAACAAGTTGATTCTCAATAAC F7 GGCTCTATACCTGCTGAACTTGGAAACTGCACCTACTTGAACAAGTTGATTCTCAATAAC Favi-9 GGCTCTATACCTGCTGAACTTGGAAACTGCACCTACTTGAACAAGTTGATTCTCAATAAC Solyc11g016930.1 GGCTCTATACCTGCTGAACTTAGAAACTGCACCTACTTGAACAAGTTGATTCTCAATAAC

967 AACAAACTATCTGGAAATATTCCACCAGAAATCTCTCAATTAACAAGGCTCAAGGTGCTC 981 AACAAACTATCTGGAAATATTCCACCAGAAATCTCTCAATTAACAAGGCTCAAGGTGCTC F5 AACAAACTATCTGGAAATATTCCACCAGAAATCTCTCAATTAACAAGGCTCAAGGTGCTC F7 AACAAACTATCTGGAAATATTCCACCAGAAATCTCTCAATTAACAAGGCTCAAGGTGCTC Favi-9 AACAAACTATCTGGAAATATTCCACCAGAAATCTCTCAATTAACAAGGCTCAAGGTGCTC Solyc11g016930.1 AACAAACTATCTGGAAATATTCCACCAGAAATCTCTCAATTAACAAGGCTCAAGGTGCTC

967 TCCTTGGCTAACAACAATCTTTCTGGTAACATACCACCATTCTCAGGATTGACTGATTTT 981 TCCTTGGCTAACAACAATCTTTCTGGTAACATACCACCATTCTCAGGATTGACTGATTTT F5 TCCTTGGCTAACAACAATCTTTCTGGTAACATACCACCGTTCTCAGGATTGACTGATTTT F7 TCCTTGGCTAACAACAATCTTTCTGGTAACATACCACCATTCTCAGGATTGACTGATTTT Favi-9 TCCTTGGCTAACAACAATCTTTCTGGTAACATACCACCATTCTCAGGATTGACTGATTTT Solyc11g016930.1 TCCTTGGCTAACAACAATCTTTCTGGTAACATACCACCATTCTCAGGATTGACTGATTTT

967 GAGTATGGAGGAAATAGACATCTCTGCGGTGGAACATTAGCCAAATGTGGTTGA 981 GAGTATGGAGGAAATAGACATCTCTGCGGTGGAACATTAGCCAAATGTGGTTGA F5 GAGTATGGAGGAAATAGACATCTCTGCGGTGGAACATTAGCCAAATGTGGTTGA

Results

F7 GAGTATGGAGGAAATAGACATCTCTGCGGTGGAACATTAGCCAAATGTGGTTGA Favi-9 GAGTATGGAGGAAATAGACATCTCTGCGGTGGAACATTAGCCAAATGTGGTTGA Solyc11g016930.1 GAGTATGGAGGAAATAGACATCTCTGCGGTGGAACATTAGCCAAATGTGGTTGA Figure 16: Nucleotide alignment of the coding sequence of the candidate gene LRR-RLP from the resistant lines (GF13 X 981 (F7), GF13 X 967 (F5) and Favi-9), susceptible lines (981, 967) compared with the genomic DNA sequence of LRR-RLP (Solyc11g016930.1) from the Sol Genomics database. The nucleotide adenine of susceptible lines (967, 981) has been replaced by the nucleotide guanine in the resistant lines (F5, F7, and Favi-9).

In addition to the SNPs that were located in the intron parts of the amplified fragments of the LRR-RLP gene, one SNP was observed in the cDNA sequence (at nucleotide position 382). The coding sequences of LRR-RLP were obtained by extracting the exon parts from the amplified fragments for each tomato line. The nucleotide adenine in position 382 of the cDNA of susceptible lines (967, 981) has been replaced by the nucleotide guanine in the resistant lines (F5, F7, and Favi-9) as shown in figure 16. Translating the coding sequences to peptides reveals that the substitution of the nucleotide A382 to G382 leads to the amino acid change from arginine (R) in susceptible lines to glycine (G) in resistant lines in position 128 (Figure 17).

1 10 20 30 40 50 60 | | | | | | | 967 MWACIKFFISQEFRSRSCECLLDYCQRLLLHNVTASNDIKCLQGLKDSFKDPNVNFNSWN 981 MWACIKFFISQEFRSRSCECLLDYCQRLLLHNVTASNDIKCLQGLKDSFKDPNVNFNSWN F5 MWACIKFFISQEFRSRSCECLLDYCQRLLLHNVTASNDIKCLQGLKDSFKDPNVNFNSWN F7 MWACIKFFISQEFRSRSCECLLDYCQRLLLHNVTASNDIKCLQGLKDSFKDPNVNFNSWN Favi-9 MWACIKFFISQEFRSRSCECLLDYCQRLLLHNVTASNDIKCLQGLKDPFKDPNVNFNSWN Solyc11g016930.1 MWACIKFFISQEFRSRSCECLLDYCQRLLLHNVTASNDIKCLQGLKDSFKDPNVNFNSWN

967 FSNYSMGFICKFVGVIYWNNLENRMISLSLPNMNLSVQLPDAFKCCSSLATLHLSGNSFS 981 FSNYSMGFICKFVGVIYWNNLENRMISLSLPNMNLSVQLPDAFKCCSSLATLHLSGNSFS F5 FSNYSMGFICKFVGVIYWNNLENRMISLSLPNMNLSVQLPDAFKCCSSLATLHLSGNSFS F7 FSNYSMGFICKFVGVIYWNNLENRMISLSLPNMNLSVQLPDAFKCCSSLATLHLSGNSFS Favi-9 FSNYSMGFICKFVGVIYWNNLENRMISLSLPNMNLSVQLPDAFKCCSSLATLHLSGNSFS Solyc11g016930.1 FSNYSMGFICKFVGVIYWNNLENRMISLSLPNMNLSVQLPDAFKCCSSLATLHLSGNSFS

967 GSIPAELRNCTYLNKLILNNNKLSGNIPPEISQLTRLKVLSLANNNLSGNIPPFSGLTDF 981 GSIPAELRNCTYLNKLILNNNKLSGNIPPEISQLTRLKVLSLANNNLSGNIPPFSGLTDF F5 GSIPAELGNCTYLNKLILNNNKLSGNIPPEISQLTRLKVLSLANNNLSGNIPPFSGLTDF F7 GSIPAELGNCTYLNKLILNNNKLSGNIPPEISQLTRLKVLSLANNNLSGNIPPFSGLTDF Favi-9 GSIPAELGNCTYLNKLILNNNKLSGNIPPEISQLTRLKVLSLANNNLSGNIPPFSGLTDF Solyc11g016930.1 GSIPAELRNCTYLNKLILNNNKLSGNIPPEISQLTRLKVLSLANNNLSGNIPPFSGLTDF

967 EYGGNRHLCGGTLAKCG* 981 EYGGNRHLCGGTLAKCG* F5 EYGGNRHLCGGTLAKCG* F7 EYGGNRHLCGGTLAKCG* Favi-9 EYGGNRHLCGGTLAKCG* Solyc11g016930.1 EYGGNRHLCGGTLAKCG* Figure 17: Amino acids alignment of LRR-RLP protein from the resistant lines (GF13 X 981 (F7), GF13 X 967 (F5) and Favi-9), susceptible lines (981, 967) compared with the polypeptide sequence of LRR-RLP (Solyc11g016930.1) from the Sol Genomics database. The amino acid arginine in susceptible lines (967, 981) is substituted to glycine in the resistant lines (F5, F7, and Favi-9).

Results

To study how this amino acid substitution affects the secondary structure of the protein LRR-RLP, online available SOPMA software (ExPasy website) was used to predict the secondary structure of LRR-RLP of the resistant and susceptible lines. The prediction showed that the change of the charged amino acid arginine to the hydrophobic amino acid glycine in resistant lines leads to a change in the secondary structure of the protein where the amino acid change is located. As it is shown in figure 18, the helix structure of susceptible lines is changed to a coil structure in resistant lines. This amino acid change is located in one of the LRR repeats of the protein.

S-lines

MWACIKFFISQEFRSRSCECLLDYCQRLLLHNVTASNDIKCLQGLKDSFKDPNVNFNSWNFSNYSMGFIC eeeeeeeeechhccccchhhhhhhhhhheeeccccccchhhhhhhhhhcccttccccttccccccheehh

KFVGVIYWNNLENRMISLSLPNMNLSVQLPDAFKCCSSLATLHLSGNSFSGSIPAELRNCTYLNKLILNN hheeeeehhhhhhhheeecccccceeeeccthhhhhhhhheeecttcccccccchhhhhhhhhhheeect

NKLSGNIPPEISQLTRLKVLSLANNNLSGNIPPFSGLTDFEYGGNRHLCGGTLAKCG tccccccccc hhhhhhheeeehhcccccccccccccccceeettcceeettehhhht

R-lines MWACIKFFISQEFRSRSCECLLDYCQRLLLHNVTASNDIKCLQGLKDSFKDPNVNFNSWNFSNYSMGFIC eeeeeeeee chhccccchhhhhhhhhhheeeccccccchhhhhhhhhhcccttccccttccccccheehh KFVGVIYWNNLENRMISLSLPNMNLSVQLPDAFKCCSSLATLHLSGNSFSGSIPAELGNCTYLNKLILNN hheeeeehhhhhhhheeecccccceeeeccthhhhhhhhheeecttccccccccccccccheehheeect

NKLSGNIPPEISQLTRLKVLSLANNNLSGNIPPFSGLTDFEYGGNRHLCGGTLAKCG tccccccccchhhhhhheeeehhcccccccccccccccceeettcceeettehhhht

Figure 18: comparison between the secondary structure of the RLP in Susceptible (S) and resistant (R) tomato lines using SOPMA software. At SNP site R128G, the helical structure of the susceptible lines is changed to the coil structure in resistant lines. Sequences of amino acids are shown in black upper rows and the predicted structures are shown in different color in the lower rows. The index of this analysis is as the following: Alpha helix: h, Extended strand: e, Beta turn: t, Random coil: c. the changed amino acids are marked with yellow background. 3.1.6 Relative expression of Receptor-like kinase (RLK) According to the analysis of microarray data, Receptor-like kinase gene (Solyc11g011880.1) was found to be the highest regulated among the differentially regulated genes. This could be clearly seen in the linear representation of the expression level of differentially regulated genes (Figure 14). In order to confirm that RLK is highly expressed in the resistant lines, three replicates of cDNA prepared from each of resistant (T3 and F7) and susceptible (981) lines were subjected to real-time quantitative PCR (qPCR) with gene

Results specific primers.cDNA used in qRT-PCR analysis were prepared from RNA samples that have been exposed to transcriptome analysis. As shown in figure 11, RLK was up-regulated in TYLCV resistant lines when compared to the expression level of RLK in TYLCV susceptible lines with a fold change of 1.4 under P value 0.027.

1,4

1,2

)

α 1

EF1

expression 0,8

0,6 (RLK/

Relative 0,4

0,2 0 R S Figure 19: Relative expression of Receptor-like kinase in TYLCV resistant lines (R) and TYLCV susceptible line (S) as analyzed via qRT-PCR. EF1α was used as reference for the RLK relative expression. RLK is significantly highly expressed in R lines in comparison to the expression of RLK in S lines (P≥0.05). 3.1.7 Conserved domains of RLK and LRR-RLP Polypeptide sequences of RLK (Solyc11g011880.1) and LRR-RLP (Solyc11g016930.1) were submitted to conserved domain database (CDD) in order to look for the organization and function of the domains of RLK and LRR-RLP. CDD analysis (Figure 20) showed that RLK belongs to the super family protein kinase; the protein has two stress-anti-fungus domains that have a role in salt stress response and also has anti-fungal activity, one catalytic domain of the Serine/Threonine kinase, Interleukin-1 Receptor Associated Kinases (STKc_IRAK) and the catalytic domain of tyrosine kinase (TyrKc). STKc catalyzes the transfer of the gamma-phosphoryl group from ATP to serine/threonine residues on protein substrates. IRAKs are involved in Toll-like receptor (TLR) and interleukin-1 (IL-1) signaling pathways, and thus are critical in regulating innate immune responses and inflammation. In addition, RLK has many sites for ATP and polypeptide binding. The protein LRR-RLP has four leucine-rich repeats.

Results

Figure 20: The domain organization of RLK (a) and LRR-RLP (b).RLK has two stress-anti- fungus domains, catalytic domain of the Serine/Threonine kinases, Interleukin-1 Receptor Associated Kinases (STKc_IRAK), tyrosine kinase catalytic domain (TyrKc). LRR-RLP has four leucine-rich repeats.

3.2 Evaluation of Potato virus Y (PVY) and Tomato spotted wilt virus (TSWV) resistance in transgenic Nicotiana tabacum SNN and Solanum lycopersicum M82 lines having single or double gene constructs 3.2.1 Tobacco and tomato plants expressing single or double genes

Tobacco and tomato plants were transformed with one of the five constructs (pRB35S:ΔCPIP1-NSm, pRB35S: ΔCPIP1-ΔM541, pRB35S:ΔM541, pRB35S:ΔCPIP1, and pRB35S: NSm) to obtain PVY and/or TSWV resistant tobacco and tomato plants. The five constructs have either single or double gene combinations of three genes; ΔCPIP1, ΔM541, and NSm. Tobacco plants were transformed with the constructs pRB35S:ΔCPIP1-NSm, pRB35S:ΔM541 and pRB35S: NSm. Regenerated tobacco plants were examined by western blot for protein expression (Figures 21 22 and 23). Western blot analysis of protein expression in plants transformed with the double constructs (pRB35S:ΔCPIP1-NSm, pRB35S: ΔCPIP1- ΔM541) will give two fragments. One large size fragment (around 70kDa) represents the detection of the two proteins ΔCPIP1-NSm or ΔCPIP1-ΔM541, and another smaller band represent either ΔM541 (30kDa) or NSm (35kDa). ΔM541 and NSm might be cleaved from the larger one during protein preparation process since they are linked together by a hyperlinker (29 aa).

Results

WT ΔCPIP1- 1 2 3 4 5 6 7 8 9 SNN NSm

70 KD 55 KD

35 KD

Figure 21: Detection of the expression of ΔCPIP1-NSm in regenerated N. tabacum. Fifty micrograms of total protein were separated by 10% SDS-PAGE and immunodetected using Anti-HA antibodies. Wild type tobacco was used as a negative control; a previously tested ΔCPIP1-NSm transgenic Microtom tomato plant was used as a positive control. Lanes 1-9 are transformed tomato plantlets. Upper fragment represents uncleaved ΔCPIP1- NSm protein; lower fragment represents the cleaved NSm protein. ΔCPIP1- WT 1 2 3 4 5 6 7 ΔM541 SNN

70 KD 55 KD

35 KD

25 KD

Figure 22: Detection of the expression of ΔM541 in regenerated N. tabacum. Fifty micrograms of total protein were separated by 10% SDS-PAGE and immunodetected using Anti-HA antibodies. Wild type SNN tobacco was used as a negative control, a previously tested ΔCPIP1- ΔM541 transgenic tobacco plant was used as a positive control. Lanes 1-7 are transformed tobacco plantlets. Upper fragment represents non cleaved ΔCPIP1- ΔM541 protein; 30kDa lower fragment represents the cleaved ΔM541 protein. NSm WT 1 2 3 4 5 6 SNN SNN

55 KD

35 KD

Figure 23: Detection of NSm expression in regenerated N. tabaccum. Fifty micrograms of total protein were separated by 10% SDS-PAGE and immunodetected using Anti-HA antibodies. Wild type SNN tobacco was used as a negative control, previously tested NSm transgenic tobacco plant was used as a positive control. Lanes 1-6 are transformed tobacco plantlets. The fragment at 35 kDa represents NSm protein.

Results

The Number of tobacco plants regenerated from each construct transformation and the number of transgenic plants obtained are summarized in table 11. Twenty-two transgenic tobacco plants were obtained for each pRB35S:ΔCPIP1-NSm and pRB35S:ΔM541, but only thirteen out of ninety-six were obtained for plants transformed with pRB35S: NSm.

Construct Name No. of regenerated No. of transgenic tobacco plantlets plantlets pRB35S:ΔCPIP1-NSm 76 22 pRB35S:ΔM541 72 22 pRB35S:NSm 96 13 Table 11: Number of regenerated tobacco plants for each construct and number of plants expressing the corresponding protein The constructs pRB35S:ΔCPIP1-NSm, pRB35S:ΔM541 and pRB35S:ΔCPIP1 were also used to transform tomato plants. Protein expression was tested in regenerated tomato plants by western blot analysis for the constructs pRB35S:ΔCPIP1-NSm and pRB35S:ΔM541 shown in figures 24 and 25, respectively. Tomato plants transformed withΔCPIP1 were tested for the presence of the transcript by reverse transcriptase PCR as shown in figure 26. Number of tomato plants regenerated from each construct and the number of transgenic plants are summarized in table 12. Eight transgenic tomato plants were obtained when tomato plants were transformed with the constructs pRB35S:ΔM541and pRB35S:ΔCPIP1. For the construct pRB35S:ΔCPIP1-NSm, seventeen transgenic plants were obtained when twenty one regenerated tomato plantlets have been tested for the expression of ΔCPIP1.

ΔCPIP1- WT 1 2 3 4 5 6 7 NSm M82

70 KD 55 KD

35 KD

Figure 24: Detection of the expression of ΔCPIP1-NSm in regenerated S. lycopersicum (cultivar M82). Fifty micrograms of total protein were separated by 10% SDS-PAGE and immunodetected using Anti-HA antibodies. Wild type M82 tomato was used as a negative control, previously tested ΔCPIP1-NSm transgenic M82 tomato plant was used as a positive control. Lanes 1-7 are transformed tomato plantlets. Upper fragment represents uncleaved ΔCPIP1-NSm protein; lower fragment represents the cleaved NSm protein.

Results

WT ΔCPIP1- 1 2 3 4 5 6 7 M82 ΔM541 70 KD

55 KD

35 KD

25 KD

Figure 25: Detection of the expression of ΔM541 in regenerated S.lycopersicum (cultivar M82). Fifty micrograms of total protein were separated by 10% SDS-PAGE and immunodetected using Anti-HA antibodies. Wild type M82 tomato was used as a negative control, previously tested ΔCPIP1- ΔM541 transgenic SNN tobacco plant was used as a positive control. Lanes 1-7 are transformed tomato plantlets. Upper fragment represents non cleaved ΔCPIP1- ΔM541 protein; lower fragment represents ΔM541 protein.

ΔCPIP1- WT pCRBlunt: M 1 2 3 4 5 6 7 8 9 10 11 NSm M82 ΔCPIP1

Figure 26: Detection of the expression of ΔCPIP1 in regenerated S. lycopersicum (cultivar M82). Ten microliters of PCR products were loaded on 1% agarose gel. The wild type M82 tomato was used as a negative control, ΔCPIP1-NSm transgenic tobacco and the plasmid pCRBlunt: ΔCPIP1 were used as positive control for this reaction, lanes 1-11 are the transformed tomato plantlets. M: Gene ruler 100 bp DNA ladder (Fermentas, Germany).

Results

Construct Name No. of regenerated No. of transgenic tomato plantlets plantlets pRB35S:ΔCPIP1-NSm 21 17 pRB35S:ΔM541 15 8 pRB35S:ΔCPIP1 16 8 Table 12: Number of regenerated tomato plants for each construct and number of transgenic plants obtained.

3.2.2 TSWV resistance in tobacco plants transformed with the single or double genes Tobacco plants that have been generated when transformed with the constructs pRB35S: pRB35S: ΔCPIP1-NSm, pRB35S: ΔM541 and pRB35S: NSm were tested for TSWV resistance via mechanical inoculation with TSWV inoculum. Two weeks after TSWV inoculation, viral symptoms (mosaic, and leaf crinkling) visibly developed on wild type tobacco plants as well as on transgenic tobacco plants expressing ΔCPIP1-NSm, ΔM541, and NSm (Figures 27, 28, and 29). Mock inoculated plants were looking healthy and did not show any virus symptoms.

Figure 27: TSWV inoculation in tobacco plants expressing ΔCPIP1-NSm. Six-eight weeks old tobacco plants were inoculated with TSWV and monitored for viral symptoms. A: mock inoculated wild type tobacco, B: TSWV-inoculated wild type tobacco, C: TSWV-inoculated ΔCPIP1-NSm transgenic plant. ΔCPIP1-NSm transgenic developed viral symptoms as they are in the wild type plants.

Figure 28: TSWV inoculation in regenerated tobacco plants expressing ΔM541. Six-eight weeks old tobacco plants were inoculated with TSWV and monitored for viral symptoms. A: mock inoculated wild type tobacco, B: TSWV-inoculated wild type tobacco, C: TSWV- inoculated ΔM541 transgenic plant. Both ΔM541 transgenic and wild type plants were showing the viral symptom.

Results

Figure 29: TSWV inoculation in regenerated tobacco plants expressing Nsm. Six-eight weeks old tobacco plants were inoculated with TSWV and monitored for viral symptoms. A: mock inoculated wild type tobacco, B: TSWV-inoculated wild type tobacco, C: TSWV-inoculated NSm transgenic plant. Tobacco plants in B and C developed symptoms. ∆CPIP1-∆M541 transgenic tobacco plants (T1) were evaluated for TSWV resistance. Symptoms resulting from TSWV mechanical inoculation have been observed in all inoculated T1 transgenic plants two weeks after inoculation, although the plants were expressing the protein ∆CPIP1-∆M541 that should confer PVY as well as TSWV resistance to these plants. Plants were tested for the expression of ∆CPIP1- ∆M541 via western blot (Figure 30). Line 4-4 Line 8-1

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 9 70 KD 70 KD

35 KD 35 KD 25 KD 25 KD

Line 17- Line 41- 1 2 3 24 5 6 7 8 1 2 3 4 4 5 6 7 8 9 9 70 KD 70 KD

35 KD 35 KD

25 KD 25 KD Figure 30: Detection of the expression of ∆CPIP1-∆M541 in T1 transgenic SNN plants. Six- eight weeks old T1 ∆CPIP1-∆M541 transgenic tobacco plants were mechanically inoculated with TSWV, monitored for viral symptoms, and then detected for ∆CPIP1-∆M541expression. Fifty micrograms of total protein were separated by 10% SDS-PAGE and immunodetected using Anti-HA antibodies. Lanes 1-7: TSWV-inoculated ∆CPIP1-∆M541 plants, 8: mock inoculated plants and 9: non-inoculated plants.

Results

3.2.3 T1 transgenic tobacco and tomato lines Seeds collected from tobacco plants expressing ΔCPIP1-NSm and NSm were germinated. DNA isolated from the germinated plants was tested for the presence of the transgenes ΔCPIP1-NSm and NSm by PCR using specific primers for each construct. Plants that showed positive results in PCR were also tested for protein expression by western blot. Number of T1 plants expressing ΔCPIP1-NSm and NSm is summarized in tables 13 and 14. Ten plants from three transgenic lines were tested. For ΔCPIP1-NSm; 6, 7, 0 plants were having the transgene and expressing the protein for the lines 21, 35, and 69 respectively. For NSm; five plants from each line 21 and 61 were positive, and this increases to 8 for line 58. Plants that showed positive results regarding transgene and protein expression were kept in the greenhouse and for the collection of T2 seed.

Line No. Number of plants tested Positive plants 21 10 6

35 10 7

69 10 0

Table 13: Number of T1 tobacco plants expressing ΔCPIP1-NSm.

Line No. Number of plants tested Positive plants 21 10 5 58 10 8 61 10 5 Table 14: Number of T1 tobacco plants expressing NSm.

Seeds of transgenic tomato plants expressing ΔCPIP1 have been selected on Kanamycin/ MS medium. Fifty seed from three ΔCPIP1 tomato lines were used for the selection. Numbers of T1 tomato plants that have been germinated for each ΔCPIP1 transformed tomato line are listed in the following table 15. Twenty-three seeds were germinated from the transgenic tomato line 11, eleven plants from line number 12, and twenty-four plants for the line 16. Germinated plants were kept in the greenhouse for seed collection.

Line No. Number of plants tested Germinated plants 11 50 23 12 50 11 16 50 24 Table 15: Number of T1 tomato plants expressing ΔCPIP1.

Results

3.3 Interaction partners of Tomato yellow leaf curl virus (TYLCV)

3.3.1 TYLCV working isolate A TYLCV isolate obtained from TYLCV infected plants that have been collected from a field experiment carried out in Jordan was sequenced to identify the isolate that was used in further analysis of this study. DNA has been isolated from TYLCV infected tomato plants and subjected to rolling circle amplification (RCA) reaction. Digestion of the RCA products by the restriction endonuclease SphI gave a linear 2.8 kb fragment in TYLCV infected tomato samples and 2.7 Kb pUC19 fragments that is provided by the manufacturer as a positive control (Figure 31A). No DNA products were observed when healthy tomato plant and the water negative control were used as a template in RCA reaction followed by SphI restriction reaction. The predicted open reading frames of the sequenced TYLCV isolate that have been used in the subsequent analysis were determined as it is shown in Figure 31B. The full genomic sequence of TYLCV (Supplement 2) has 99% identity with the Jordanian mild isolate of Tomato yellow leaf curl virus-Mild (GenBank: EF158044.1).

Figure 31: (A): SphI digestion of RCA products in TYLCV infected samples (1-4), pUC19 vector, H: healthy tomato plant, - ve: negative control, M: Generuler 1Kb ladder. (B): Sequenced TYLCV genome with its six overlapping open reading frames. V1: TYLCV coat protein, V2: movement protein, C1: replication initiation protein, C2: transcription activator protein, C3: replication enhancer protein, C4: determinant of symptom expression.

Results

3.3.2 Subcellular localization of V1, V2, and C2 In order to study the localization of TYLCV proteins (V1, V2, and C2), the open reading frames of these genes were amplified by PCR using specific primers for each gene, cloned into Gateway entry vector pENTR/D-TOPO and confirmed by sequencing. These genes were transferred into the expression vectorspK7FWG2 (V1), pK7WGF2 (V2, C2) pGWB661 (V2, C2) carrying GFP. TYLCV ORFs cloned into an expression vector are shown in figures 32 (a, b, c) and 33 (a, b). Agrobacterium mediated expression of the TYLCV ORFs- GFP constructs was analyzed using confocal laser scanning microscope (CLSM) two days after agro-infiltration of N. benthamiana leaves.Images obtained in CLSM analysis showed that V1 is localized in the cell nucleus (Figure 32d), while V2 is localized in the cytoplasm and cell periphery as it is shown in figures 32e and. 33d. Figures 32f and 33c indicated the nuclear localization of C2 when it is fused with GFP and RFP respectively.

a P35S V1 GFP T35S

b P35S GFP V2 T35S

c P35S GFP C2 T35S

d e f

Figure 32: GFP-fusion constructs and their transient expression in N. benthamiana. Graphical representation of C-terminal GFP fusion protein for ORF V1 (a), and N-terminal GFP fusion protein of ORF V2 and C2 (b, c). CLSM images of N. benthamiana cells transiently expressing V1-GFP and GFP-C2 showed that V1 (d) and C2 (f) are localized in the nuclei, while GFP-V2 is localized in the cytoplasm and periphery (e). Scale bar: 10µm.

Results

a P35 RFP C2 NOS S

b P35 RFP V2 NOS S c d

Figure 33: RFP-fusion constructs and their transient expression in N.benthamiana. Graphical representation of the N-terminal RFP fusion protein of ORF C2 (a) and V2 (b). CLSM images of N. benthamiana cells transiently expressing RFP-C2 and RFP-V2 showed that C2 (c) is localized in the nucleus, while RFP-V2 is localized in the cytoplasm and periphery (d). Scale bar: 10µm.

3.3.3 Candidate interaction partners of TYLCV ORFs To identify host factors that have a role in establishing successful TYLCV infection, Nano LC-Mass spectrometry (MS) has been used. N. benthamiana plant samples that showed the expression of GFP and RFP fused protein of V1, V2 and C2 were subjected to immunoprecipitation (IP) to co-precipitate TYLCV viral protein with host proteins in the proximity. Three replicates of co-IP proteins of GFP fusion protein of V1, V2, C2 and RFP fusion protein of V2 and C2 were digested by trypsin to be analyzed in MS. The raw MS data of proteins that have been detected with high confidence in the tested samples were searched against N. benthamiana database, this leads to the identification of some candidate proteins that might interact with TYLCV viral proteins and play a role in the TYLCV infection process (Table 16).

Results

Pulled Description of the protein detected in co-IP Accession ΣCoverage No. of Peptide protein proteins score V1-GFP ATP synthase subunit gamma Niben101Scf00033g01001.1 11.21 1 0.85 Hop-interacting protein/ Putative E3 ubiquitin-protein Niben101Scf10388g01003.1 2.25 3 1.45 ligase GFP-V2 Heat shock protein Hsp90 family Niben101Scf06684g01005.1 3.87 8 2.80 GTPase Era (Small GTP-binding protein domain) Niben101Scf08670g00031.1 1.64 2 1.68 Tubulin alpha-1 chain-like Niben101Scf23114g00002.1 7.35 11 4.0 Heat shock protein 70kD Niben101Scf07275g02012.1 7.10 12 2.26 GFP-C2 Heat shock protein 70kD Niben101Scf07275g02012.1 7.10 12 2.26 Heat shock protein Hsp90 family Niben101Scf06684g01005.1 3.87 8 2.8

RFP-V2 SAUR-like auxin-responsive protein family Niben101Scf06413g02006.1 7.02 4 0.87 NB-ARC domain-containing disease resistance protein Niben101Scf07728g01020.1 1.92 1 0.20 RFP-C2 U4/U6 small nuclear ribonucleoprotein Prp3 (Pre- Niben101Scf07227g02008.1 5.51 2 0.54 mRNA-splicing factor 3) Table 16: Proteins detected in MS for ORFs (V1, V2, and C2) of TYLCV genome.Accessions refer to N. benthamiana sol genomics database, Number of peptides detected for each protein is one for all evaluations, and peptide score represents Xcorr value for each protein.

Discussion

4. Discussion

4.1 Gene expression does not respond to treatments Transcriptome analysis performed for the samples collected from plants grown in different environmental conditions in the field experiment indicated that the clustering of the samples is not influenced by the environmental conditions where plants were grown. In some clusters, samples from contradicted treatments (healthy vs. infected, hot vs. cold, watered vs. drought) were grouped together. This unexpected clustering pattern might refer to different factors. One factor could be the slight difference in temperature between both sites of the experiment; this could be noticed from the average temperature in Homra (44.8oC) and in the University (41.5oC). The temperatures 44.8oC and 41.5oC were stressful for tomato at both sites not only in Homra field, since Rivero et al., (2001) reported that heat stress in tomato could be achieved at 35oC. Another point that may explain that clustering pattern is the manifestation of whiteflies in the field during the experiment, although the fields were sprayed regularly with insecticides. These whiteflies may carry other viruses or they might feed on TYLCV-inoculated plants and transfer virus particles to the non-inoculated healthy plants when they feed on them. This could be supported by the data of symptoms evaluation represented by disease severity index (DSI) in the tables 6 and 7. DSI data indicate that many healthy plants showed a variable degree of TYLCV disease symptoms even for TYLCV resistant lines in addition to those lines that were not inoculated with TYLCV at the beginning of the experiment. A large number of healthy 981 susceptible plants were showing high values of DSI (DSI values 3 and 4); plants from these lines not even survived till the end of the experiment. Plants of the resistant lines have a DSI value more than zero or one, and in some lines, DSI values is 4. High DSI values mean that the plants were suffering from the high stress of viral infection . In this study, differentially expressed genes have been grouped only according to the genotype. This means that genes have different expression profile between TYLCV resistant and TYLCV susceptible lines. So the transcriptome profile resulted from this experiment should explain how resistant and susceptible lines respond differently to the TYLCV challenge regardless the abiotic stress that the plants were facing. Two genes (RLK and LRR- RLP) among those that have been differentially expressed might contribute to TYLCV resistance since it is reported that RLKs and RLPs have a role in disease resistance in some plant species (Liebrand et al., 2013; Belfanti et al., 2004; Zhang et al., 2013b). Chen et al.

Discussion

(2013) found that 82.5% of differentially regulated receptor (-like) kinase genes were up- regulated in the TYLCV resistant line CLN2777A when compared to the susceptible line TMXA48-4-0 . Other genes that showed overexpression in resistant lines are not belonging to the gene families that encode for proteins that are known to be involved in viral resistance. But those genes probably have an indirect role in defence mechanism in one way or another since TYLCV resistance is a result of gene networks that are triggered by a biochemical stimulus of virus inoculation (Genoud and Metraux 1999). Eybishtz et al. (2009) found that when they silenced the Permease I-like protein gene in TYLCV resistant lines through tobacco rattle virus (TRV)-based silencing, resistant tomato plants showed a susceptible phenotype and resistance collapse when they were inoculated with TYLCV. Permease I-like protein gene was found to be up regulated in resistant lines when compared to susceptible ones. Eybishtz et al. (2009) indicated that it is not shown in the literature any expected role of Permease I-like protein gene in the resistance of plants to a pathogen or in virus-host interactions.

4.2 LRR-RLP interacts with RLK to initiate signaling and mediates resistance response to TYLCV in resistant lines Plants are using a wide range of receptors at the cell surface and within the cell to detect and defend themselves against pathogens (Gomez –Gomez and Boller, 2002). RLKs and RLPs are among the five typical protein families that were defined as pathogen recognition molecules (Andolfo et al., 2013); other families are Toll/Interleukin-1 Receptor- Nucleotide-Binding Site- Leucine-Rich Repeats (TIR-NBS-LRRs), Coiled Coil-Nucleotide- Nucleotide Binding Site- Leucine-Rich Repeats (CC-NBS-LRRs), and the kinase-like proteins (Andolfo et al., 2013). RLPs and RLKs are involved in plant defence in addition to different biological processes such as development and cell differentiation (Yang et al., 2012). In their analysis of the chromosomal locations of candidate pathogen recognition genes in tomato, Andolfo and colleagues (2013) considered the RLP (Solyc11g016930.1) and RLK (Solyc11g011880.1) as part of the pathogen recognition mechanism of tomato plants. These receptors RLP (Solyc11g016930.1) and RLK (Solyc11g011880.1) were up-regulated in TYLCV resistant tomato lines when compared to susceptible lines according to the microarray analysis performed in this study. Many RLPs have been recognized and functionally identified in different plant species as a resistance gene or as pathogen recognition receptors to several pathogens. In tomato, the

Discussion

RLPs (Cf-2, Cf-4, Cf4E, Cf-5, and Cf-9B) are involved in the resistance to Cladosporium fulvum (Dixon et al., 1996; Takken et al., 1999; Panter et al., 2002; Rooney et al., 2005; Liebrand et al., 2013). Apple plants have the gene HcrVF2 which encodes an RLP that confers apple resistance to Venturia inequalis (Belfanti et al., 2004). The Arabidopsis RLP1, RLP30, and RLP42 are pathogen recognition receptors to Xanthomonas spp., Sclerotinia sclerotioum and for the fungal endopolygalacturonases respectively (Jehle et al., 2013; Zhang et al., 2013a & b). RLP-type receptors are working together with RLKs to mediate signaling and deliver the message since RLPs have an extracellular LRR domain and a C-terminal membrane anchor but lack the cytoplasmic kinase domain (Tör et al., 2009). The RLKs CLAVATA and ERECTA are working together with the RLPs CLV2 and TMM to transmit the signal from the extracellular matrix to the intracellular space (Waites and Simon, 2000; Shpak et al., 2005). In addition to Arabidopsis, it is reported that the association of the tomato LRR-RLK SOBIR1 with various RLPs (Ve1, EIX1, and Cf-4) has an important role in RLP-mediated resistance to fungal pathogens (Liebrand et al., 2013; Fradin et al., 2014). Gust and Felix (2014) assumed that RLPs associated with SOBIR1 are equivalent in structure and function to the typical receptor kinases. The domain organization of the LRR-RLP and the RLK (Figure 20) showed that RLP is composed of four leucine rich repeats (LRR) and lacking any cytoplasmic kinase domain, while RLK has a catalytic domain of the Serine/Threonine kinases, Interleukin-1 receptor associated kinases and related STKs (STKc_IRAK) but it lacks an extracellular LRR domain. LRR domain is thought to mediate ligand perception (Kobe and Kajava, 2001). Association of the RLP (Solyc11g016930.1) with the RLK (Solyc11g011880.1) would result in a complex that has an extracellular LRR domain with a cytoplasmic kinase domain, thus will provide protein structures that are required to receive a signal, mediate a message and initiate an immune response to any trigger, e.g. TYLCV. Gust and Felix (2014) proposed a model for the activation process of some RLPs and RLKs. They postulated that RLPs are associated to the adaptor RLKs, this association is important for the proper localization, stability, and function of RLPs and RLKs. The association of these receptors could occur through the LRR domain, an ionic interaction of the oppositely charged juxtamembrane domains, or by a helix-helix interaction of the transmembrane domains. Guozhi et al., (2015) indicate that the dimerization motif GxxxG (G is Glycine; x is any amino acid) in the transmembrane domain of RLK SOBIR1 is necessary for the complex formation with RLP. The RLP/Adaptor is equivalent to RLKs, will undergo

Discussion complex formation with co-receptors that bring the cytoplasmic domains of the partners on proximity allowing phosphorylation and cytoplasmic signal output (Gust and Felix, 2014). Since RLPs and RLKs are associating together, it is suggested that the up-regulated LRR-RLP (Solyc11g016930.1) and RLK (Solyc11g011880.1) are cooperating together as most of the receptors from their protein families are working together, and RLP-RLK association could directly or indirectly recognize TYLCV as a pathogen. So, the TYLCV resistant tomato lines could start the downstream signaling and defend themselves against the pathogen. Tables 6 and 7 which summarize the symptom evaluation of the plant in this experiment showed that plants of TYLCV resistant lines showed better performance than those plants of TYLCV susceptible lines. This could be indicated by the number of plants showing TYLCV symptoms of different disease severity.

4.3 Resistant lines are distinguished from susceptible lines via SNPs detected in LRR-RLP The sequencing analysis of the amplified fragments of the LRR-RLP gene showed ten SNPs that are conserved in resistant lines when compared to the susceptible ones. SNPs that were detected in the LRR-RLP amplified fragments from resistant and susceptible lines are located in the intron parts, while one SNP was found in the coding region of the gene. SNPs detected in LRR-RLP gene might be used to differentiate between TYLCV resistant and susceptible lines as reported by qqqq

4.4 A single amino acid change may alter the recognition specificity of the receptor like protein An SNP that is of greater importance in this study is the one that is located in the coding sequence of the LRR-RLP gene. The SNP leads to an arginine to glycine amino acid change in the LRR region of the protein that changed the secondary structure of LRR-RLP from helix structure in susceptible line to the coiled structure in resistant lines. LRRs are found in a large number of proteins with variable structure, localization and function in bacteria, fungi, plants and animals (Kobe and Kajava, 2001). It is assumed that the major role is to facilitate protein- protein interaction (Kobe and Kajave, 2001) or to bind pathogen-derived ligands (Warren et al., 1998). Many studies indicated that single amino acid changes within the LRR region of resistance proteins render the resistant plant into the susceptible phenotype, this change in the amino acid sequence either affecting recognition specificity or suppression of resistance genes

Discussion

(Halterman and Wise, (2004); Bent et al., (1994); Shen et al., (2003); Grant et al., (1995)). Warren et al., (1998) reported that a mutation that results in glutamate to lysine in the LRR region of RPS5 compromised the function of many R genes that have a role in the resistance of Arabidopsis plants to bacteria and downy mildew . According to these findings, it is of great importance to investigate if the resistance of tomato plants to TYLCV is influenced when a single amino acid is changed as what obtained in this study; when the amino acid glycine replaces arginine in TYLCV resistant lines.

4.5 ∆M541 and NSm don´t confer tobacco plants resistance to TSWV The mutant version (lacks 68 amino acids from N-terminus) of the PVY coat protein interaction partner (∆CPIP1), TSWV movement protein interaction partner (∆M541) and TSWV movement protein were used to transform tobacco and tomato plants to get resistance to PVY and TSWV respectively. Hafren et al. (2010) proposed that the role of CPIP1 in PVY infection cycle is HSP70 dependent, CPIP1 is a DnaJ protein which is assumed to function as co-chaperone and regulator of HSP70 by activating the ATPase activity of HSP70 when it interacts with the DnaJ domain of CPIP. Tobacco plants were transformed with a mutated CPIP1 lacking 65 amino acids from the N-terminus and challenged with PVY as described by Hofius et al., (2007). ∆CPIP1 used for tobacco and tomato transformation in this study lacks 68 amino acids of the CPIP1; the N-terminal deletion of 68 amino acids is a deletion of the DnaJ domain where the HSP70 interaction site is located. Tobacco or tomato transformation with ∆CPIP1 has been performed to detect the role of CPIP1 in the infection cycle of PVY, and to detect if CPIP1 is HSP70 dependent since the mutation is located in HSP70 interaction site. PVY resistance in tobacco plants transformed with ∆CPIP1 has been tested previously by Ursula Hoja and revealed that transformed tobacco plants gain resistance against PVY (Personal communication). Regarding NtDnaJ_M541, Soellick et al. (2000) postulated that the process in which NtDnaJ_M541 interacts with the movement protein NSm and facilitates viral movement is either an HSP70 dependent process or that NtDnaJ_M541is the motive force for the translocation of RNA-protein transport complex to the plasmodesmata. To proof the postulation that the movement process is mediated by HSP70, 70 amino acids spanning the DnaJ domain of NtDnaJ_M541 (HSP40) were deleted to get ∆M541 and used for tobacco transformation. Transgenic expression of ∆M541gene is expected to hinder the TSWV MP action and so the infection process of the virus is not completed.

Discussion

Unfortunately, none of the ∆M541transgenic plants were resistant to TSWV, since all tested plants were developing disease symptoms (Figure 28) when the Plants have been challenged with the virus. It is possible that the mode of action of NtDnaJ_M541 is independent of HSP70 activity, or it could be that the expression level of the endogenous NtDnaJ_M541 was higher than that of ∆M541 and for this TSWV movement is not influenced by the expression of ∆M541. Tobacco plants expressing the movement protein of TSWV (NSm) either in single (pRB 35S: NSm) or double gene constructs (pRB35S: ΔCPIP1-NSm) were showing disease symptoms (Figures 27, 29) when the transgenic plants have been challenged with TSWV. Many virus-derived gene sequences such as coat protein sequence were used to obtain virus resistant plants (Hackland et al., 1994). Coat protein mediated resistance has been obtained when the wild type CP genes were expressed, but it is also possible to get viral resistance when dysfunctional versions of viral genes are used to transform plants (Baulcombe, 1996). Lapidot et al., (1993) illustrated that movement protein-mediated protection to tobacco mosaic virus (TMV) had been achieved when a negative mutant (deleting nucleotides from the wild type sequence) of MP transgene was used to transform N. tabacum plants. Lapidot et al., (1993) proposed that the dysfunctional mutant TMV MP competes with the wild type MP for plasmodesmatal binding sites. In another study performed by Gafny et al. (1992), nine nucleotides were deleted from the cDNA of TMV MP which resulted in the deletion of three amino acids of viral MP; the amino acids change still enabled the virus to replicate but the protein lost its movement activity. Zhen-Chen et al., (1999) studied the cucumber mosaic virus (CMV) resistance of N. tabacum plants transformed with either full length or deletion mutants of CMV MP, the inoculation of regenerated plants with CMV indicates that tobacco plants transformed with the deletion mutant of CMV MP were showing higher resistance to CMV than those transformed with the full-length gene of MP.

4.6 TYLCV proteins interact with host factors Co-immunoprecipitation (co-IP) and mass spectrometry (MS) used in this study identified proteins that are expected to be recruited by TYLCV to establish a successful virus infection. The identified candidates might interact directly or indirectly with TYLCV proteins. Some clues about the potential interaction of the host factors that have been detected by MS with the viral proteins of TYLCV will be discussed in this part of this work. GTPase Era-like protein is co-precipitated with GFP-V2; V2 encodes the movement protein of TYLCV. So it is expected that GTPase has a role in the movement of TYLCV.

Discussion

TYLCV is a monopartite geminivirus that has a coat protein (encoded by the ORF V1) which functions like the NSP of the bipartite begomoviruses (Rojas et al., 2001). Carvalho et al., (2008a) characterized an NSP-interacting GTPase (NIG) that acts as a helper factor in the nucleocytoplasmic trafficking of viral DNA. NIG is a GTP binding protein that has GTPase activity, a common feature of regulatory proteins involved in protein trafficking. Carvalho et al., (2008a) proposed that NIG mediates the movement of NSP into the cytosol, then the NSP-DNA complex moves to the cell periphery through its interaction with the MP as it is reported by Sanderfoot and Lazarowitz (1995). Carvalho et al., (2008b) proposed a model for the viral DNA movement. The model suggests that the transfer of NSP- DNA complex to the MP is enabled via a mechanism that involves NIG-catalyzed GTP hydrolysis. Another proposal based on this model suggested that NIG facilitated the interaction of MP with NSP-DNA complex that moves through plasmodesmata, which moves the viral DNA into the nucleus of the next cell. Taking all these data together, it is postulated that V2 interacts with GTPase-V1-Viral DNA complex to mediate cell-to-cell movement of TYLCV DNA. Small auxin up RNA (SAUR)-like auxin-responsive protein was also detected via co- immunoprecipitation (co-IP) in V2 (MP) preparation. Although auxins are known to function in plant growth and development, they were recently found to have a role in plant-pathogen interactions (Denance et al., 2013; Pieterse et al., 2009; Santner et al., 2009). Auxin is known to be involved in disease susceptibility to viral pathogens (Spaepen and Vanderleyden, (2011); Culver and Padmanabhan, (2007)). SAUR proteins may play a role in an auxin signal transduction pathway (Hagen and Guilfoyle, 2002); they are known to have a negative impact on Auxin synthesis in Arabidopsis (Kong et al., 2013). Smith et al. (1968) reported that a reduced auxin response is a general behavior of begomovirus infection. It is reported by Padmanabhan et al. (2005, 2008) that auxin response factors have a role in the replication and movement of some viruses like TMV. This data support that the interaction of the SAUR-like auxin-responsive protein with TYLCV movement protein which is encoded by the ORF V2 has a role in the viral movement during TYLCV infection. Heat shock protein70 (HSP70) and HSP90 were expected to interact with TYLCV ORFs V2 and C2, as they were detected in mass spectrometry analysis of the co- immunoprecipitated proteins of V2 and C2. HSP70s and HSP90s are molecular chaperones that play an important role in a variety of cellular processes such as protein folding, protein refolding and aggregation, stress response, protein trafficking and signal transduction (Su and Li, 2008; Sung et al., 2001; Richter and Buchner, 2001; Pratt et al., 2008). The co-chaperone

Discussion

HOP (HSP70-HSP90 organizing protein) links the chaperoning machinery of HSP70 and HSP90 to facilitate the cellular processes which HSP70 and HSP90 are involved in (Richter and Buchner, 2001). Hop interacting protein; an Arabidopsis putative E3 ubiquitin-protein ligase homolog was co-precipitated with V1 ORF according to MS results. E3 ubiquitin- protein ligase is involved in the ubiquitination process of proteins (Mazzucotelli et al., 2006). HSP70, HSP90, and Hop (E3 ligase) were found to interact with the movement protein (encoded by V2), transcriptional activator protein (encoded by C2), and the coat protein (encoded by V1) of TYLCV genome. The expected involvement of these candidates in the movement, transcription, and assembly of TYLCV indicates the important role of chaperone and ubiquitination activities in TYLCV infection cycle. In general, HSP70 and HSP90 have an important role in the quality control of cellular proteins (Nollen and Morimoto, 2002). During virus infection, viral protein as well as host cellular proteins are regulated by the machinery of quality control (Verchot, 2012). The model proposed by Hafren et al. (2010) describes the role of the co-chaperone CPIP together with HSP70 in regulating the function of PVY CP. CPIP recruits PVY CP to HSP70 in a process that facilitates the ubiquitination and degradation of the CP. The accumulation of CP blocks virion assembly and suppresses viral genome translation, and this will hinder the cell-to-cell movement of the virus. In the same way, the Hop interaction partner which is E3 ubiquitin ligase might interact with TYLCV CP to aid in the unpacking of the virus, and hence the translation of viral proteins, cell-to-cell movement, and successful TYLCV infection. A study conducted by Gorovits et al. (2013a) showed that the inactivation of HSP70 decreases the level of TYLCV DNA indicating that HSP70 has a role in the multiplication of TYLCV in planta. The same study revealed that the intracellular movement of TYLCV CP is facilitated by HSP70 . To achieve successful infection, geminiviruses are known to change the machinery of ubiquitin and ubiquitin-like protein (reviewed by Hanley-Bowdoin et al., 2013). So, it is possible that TYLCV V1 interacts with E3 ubiquitin ligase to establish the infection. Alpha and beta tubulin proteins are subunits of microtubules; microtubules are constituents of the plant cytoskeleton (Brierley et al., 1995). Tubulin alpha-1 chain-like protein was found co-precipitated with V2 (MP); this indicates the role of the cytoskeleton in the movement of plant viruses. It is reported that microtubules play an important role in the intracellular movement of viral proteins either for animal or plant viruses (Dohner and Sodeik, 2002; Lellis et al., 2002; Harries et al., 2010; Nieh et al., 2013). During viral infection, viruses induce the formation of some structures in the infected cell such as replication complexes,

Discussion virus factories, and inclusion bodies or aggregates. The formation of these structures depends on trafficking of host and viral proteins along the cytoskeleton (Moshe et al., 2015). V2 was immuno-detected in cytoplasmic insoluble aggregates during TYLCV infection, the aggregates are increasing in size and then localized in the nucleus with the progress of the infection (Moshe et al., 2015). Gorovits et al., (2013b) reported that the large nuclear aggregates represent virus factories and also contain proteins from viral and host origin and aggregates formation indicated the plant susceptibility to the virus and a successful TYLCV infection. Moshe et al. (2015) postulate that V2 aggregates enhance the nuclear transport of TYLCV ssDNA either alone or coupled with the CP, and showed that the formation of V2 aggregates is prevented or impaired upon the disruption of cytoskeleton when they used herbicide that is known to depolymerize the microtubules. Collectively, the interaction of V2 and tubulin protein is postulated to influence the viral DNA movement during TYLCV infection. ATP synthase subunit gamma is detected as candidate interaction partners of TYLCV V1 (CP). ATP synthase generates energy to be used by the cell during ATP synthesis (Moshe et al., 2012). ATP synthase might interact with CP of TYLCV when energy is needed for the nucleocytoplasmic trafficking of viral DNA-CP complex. A protein detected in the pull-down with V2 is NB-ARC domain-containing disease resistance protein. NB-ARC domain is a nucleotide binding domain that is found in most resistance proteins, it is a functional ATPase domain that is proposed to regulate resistance (R) protein activity through its nucleotide binding site (Ooijen et al., 2008). Plant disease resistance proteins recognize pathogens by detecting their effector proteins (Martin et al., 2003). So the interaction between NB-ARC domain-containing disease resistance proteins and V2 might be the pathogen recognition step that initiates immune response against TYLCV not to facilitate TYLCV infection. U4/U6 small nuclear ribonucleoprotein Prp3 (Pre-mRNA-splicing factor 3) was co- immunoprecipitated with TYLCV C2 as indicated in MS analysis. Spliceosome, a multi ribonucleoprotein (RNP) complex catalyzes nuclear pre-mRNA splicing (Will and Lührmann, 2011). Major spliceosome in plants is composed of five types of U-rich small nuclear RNAs (snRNAs), referred to as U1, U2, U4, U5, and U6 (Lorkovic et al. 2000). For the spliceosome formation, U6 and U4 snRNAs are base-paired with each other to form U4/U6 di-snRNP which combined with U5 to form U4/U6.U5 spliceosomal tri-RNP (Will and Lührmann, 2011). The interaction of TYLCV C2 protein which is a transcription activator with U4/U6 small nuclear ribonucleoprotein might indicate that the transcriptional activity of C2 is

Discussion interfering the mRNA splicing of the host genes, so TYLCV makes use of this in the transcription of its proteins. The postulated interaction of the detected proteins with TYLCV proteins should be further proven by other methods such as yeast two hybrid system or bimolecular fluorescent complementation. When the interaction is proofed, virus induced gene silencing (VIGS) could be used to study how silencing these factors would influence TYLCV infection process. The important function of the detected host factors during viral infection should be studied in details exploiting some proteomic approaches.

List of abbreviation

5. List of abbreviation

ABA Abscisic acid At Arabidopsis thaliana Bp Base pair BR Brassinosteroids CaCV Capsicum chlorosis virus CaMV Cauliflower Mosaic virus CC Coiled coil cDNA Complementary DNA CI Cylindrical inclusion CK Cytokinin CP Capsid protein CPIP Capsid protein interaction partner oC Celsius degree Da Dalton DAMP Damage associated molecular patterns DSI Disease Severity Index eIF Eukaryotic initiation factor ET Ethylene FC Field Capacity GA Gibberellin HC-Pro Helper component- proteinase HSP Heat Shock Protein IR Intergenic region JA Jasmonic acid LRR Leucine rich repeat LysM Lysine motif MAPKs Microbe associated molecular patterns MAS Marker assisted selection min Minute ml Milliliter MP Movement protein µl Microliter NB Nucleotide binding NI Nuclear inclusion NSP Nuclear shuttle protein Nt Nicotiana tabaccum ORF Open reading frame PAMP Pathogen associated molecular patterns PD Plasmodesmata

List of abbreviation

PDR Pathogen derived resistance PepMV Pepino mosaic virus PGIPs Polygalactouranase-inhibiting proteins PIPO Pretty Interesting Potyviridae ORF PR Pathogenesis related PR5K Pathogenesis related 5-like kinase PRRs Pathogen recognition receptors PTGS Posttranscriptional gene silencing PVY Potato virus Y PWP Permanent wilting point PZSV Pelargonium zonate spot virus RCC Regulator of chromatin condensation RdRp RNA dependent RNA polymerase REn Replication Enhancer Rep Replication preotein RLCK Receptor like cytoplasmic kinase RLK Receptor like kinase RLP Receptor like protein ROS Reactive oxygen species SA Salicylic acid SAUR Small auxin up RNA sec Second Ser/Thr Serine/Threonine siRNA Short interfering RNA SNP Single nucleotide polymorphism TF Transcription factor TICV Tomato infectious chlorosis virus TIR Toll/interlukin receptor TMM Too many mouth TMV Tobacco mosaic virus TNFR Tumor necrotic factor receptor TNSV Tomato necrotic spot virus ToCV Tomato chlorosis virus ToMarV Tomato marchitez virus ToTV Tomato torrado virus TrAP Transcription activator protein TSWV Tomato spotted wilt virus TYLCV Tomato yellow leaf curl virus TYRV Tomato yellow ring virus TZSV Tomato zonate spot virus VPg Viral genome linked protein WAK Wall associated kinase

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Appendix

7. Appendix - Nucleotide alignment the genomic DNA amplified PCR product of the candidate gene Erwinina Induced protein 2 from the resistant lines (GF13 X 981 (F7), GF13 X 967 (F5) and Favi-9), susceptible lines (981, 967) compared the genomic DNA sequence of Erwinina Induced protein 2 (Solyc11g011010.1). 1 10 20 30 40 50 60 | | | | | | | 967 ATGGCGTTGGAATGGGTTGTTCTCGGCTACGCCGCCGGAGCCGAAGCCATCATGCTGCTT 981 ATGGCGTTGGAATGGGTTGTTCTCGGCTACGCCGCCGGAGCCGAAGCCATCATGCTGCTT F5 ATGGCGTTGGAATGGGTTGTTCTCGGCTACGCCGCCGGAGCCGAAGCCATCATGCTGCTT F7 ATGGCGTTGGAATGGGTTGTTCTCGGCTACGCCGCCGGAGCCGAAGCCATCATGCTGCTT Favi-9 ATGGCGTTGGAATGGGTTGTTCTCGGCTACGCCGCCGGAGCCGAAGCCATCATGCTGCTT Solyc11g011010.1 ATGGCGTTGGAATGGGTTGTTCTCGGCTACGCCGCCGGAGCCGAAGCCATCATGCTGCTT

967 CTATTAACGGTTCCGGGTCTTAACCCGCTTCGAAAAGGGCTAATTTCCGTGACCCGGAAT 981 CTATTAACGGTTCCGGGTCTTAACCCGCTTCGAAAAGGGCTAATTTCCGTGACCCGGAAT F5 CTATTAACGGTTCCGGGTCTTAACCCGCTTCGAAAAGGGCTAATTTCCGTGACCCGGAAT F7 CTATTAACGGTTCCGGGTCTTAACCCGCTTCGAAAAGGGCTAATTTCCGTGACCCGGAAT Favi-9 CTATTAACGGTTCCGGGTCTTAACCCGCTTCGAAAAGGGCTAATTTCCGTGACCCGGAAT Solyc11g011010.1 CTATTAACGGTTCCGGGTCTTAACCCGCTTCGAAAAGGGCTAATTTCCGTGACCCGGAAT

967 CTCCTCAAGCCATTTCTCTCCATTGTTCCCTTTTGCCTCTTTCTCTTAATGGATATCTAC 981 CTCCTCAAGCCATTTCTCTCCATTGTTCCCTTTTGCCTCTTTCTCTTAATGGATATCTAC F5 CTCCTCAAGCCATTTCTCTCCATTGTTCCCTTTTGCCTCTTTCTCTTAATGGATATCTAC F7 CTCCTCAAGCCATTTCTCTCCATTGTTCCCTTTTGCCTCTTTCTCTTAATGGATATCTAC Favi-9 CTCCTCAAGCCATTTCTCTCCATTGTTCCCTTTTGCCTCTTTCTCTTAATGGATATCTAC Solyc11g011010.1 CTCCTCAAGCCATTTCTCTCCATTGTTCCCTTTTGCCTCTTTCTCTTAATGGATATCTAC

967 TGGAAATACGAGACCCGACCCACTTGTGAATCCGAATCGTGTTCTCCATCGGAGTACCTT 981 TGGAAATACGAGACCCGACCCACTTGTGAATCCGAATCGTGTTCTCCATCGGAGTACCTT F5 TGGAAATACGAGACCCGACCCACTTGTGAATCCGAATCGTGTTCTCCATCGGAGTACCTT F7 TGGAAATACGAGACCCGACCCACTTGTGAATCCGAATCGTGTTCTCCATCGGAGTACCTT Favi-9 TGGAAATACGAGACCCGACCCACTTGTGAATCCGAATCGTGTTCTCCATCGGAGTACCTT Solyc11g011010.1 TGGAAATACGAGACCCGACCCACTTGTGAATCCGAATCGTGTTCTCCATCGGAGTACCTT

967 CGTCATCAGAAATCAATCATGAAATCGCAGCGTAATGCGCTTCTAATCGCCTGTGCGCTG 981 CGTCATCAGAAATCAATCATGAAATCGCAGCGTAATGCGCTTCTAATCGCCTGTGCGCTG F5 CGTCATCAGAAATCAATCATGAAATCGCAGCGTAATGCGCTTCTAATCGCCTGTGCGCTG F7 CGTCATCAGAAATCAATCATGAAATCGCAGCGTAATGCGCTTCTAATCGCCTGTGCGCTG Favi-9 CGTCATCAGAAATCAATCATGAAATCGCAGCGTAATGCGCTTCTAATCGCCTGTGCGCTG Solyc11g011010.1 CGTCATCAGAAATCAATCATGAAATCGCAGCGTAATGCGCTTCTAATCGCCTGTGCGCTG

967 GTGTTCTATTGGTTATTATACTCTGTTACTGGCCTTGTTGTGAAAGTTGAACAGCTGAAT 981 GTGTTCTATTGGTTATTATACTCTGTTACTGGCCTTGTTGTGAAAGTTGAACAGCTGAAT F5 GTGTTCTATTGGTTATTATACTCTGTTACTGGCCTTGTTGTGAAAGTTGAACAGCTGAAT F7 GTGTTCTATTGGTTATTATACTCTGTTACTGGCCTTGTTGTGAAAGTTGAACAGCCGAAT Favi-9 ´ GTGTTCTATTGGTTATTATACTCTGTTACTGGCCTTGTTGTGAAAGTTGAACAGCCGAAT Solyc11g011010.1 GTGTTCTATTGGTTATTATACTCTGTTACTGGCCTTGTTGTGAAAGTTGAACAGCTGAAT

967 AAGCGGGTGGAGAAGATGAAGGCTTCGGATTGA 981 AAGCGGGTGGAGAAGATGAAGGCTTCGGATTGA F5 AAGCGGGTGGAGAAGATGAAGGCTTCGGATTGA F7 AAGCCCGTGGAGAAGATGAAGGCTTCGGATTGA Favi-9 AAGCGGGTGGAGAAGATGAAGGCTTCGGATTGA Solyc11g011010.1 AAGCGGGTGGAGAAGATGAAGGCTTCGGATTGA

Appendix

- Nucleotide alignment the genomic DNA amplified PCR product of the candidate gene Splicing factor from the resistant lines (GF13 X 981 (F7), GF13 X 967 (F5) and Favi-9), susceptible lines (981, 967) compared the genomic DNA sequence of splicing factor Solyc11g039950.1.

1 10 20 30 40 50 60 | | | | | | | 967 ATGGCGGAGAACCCTAATTGCACTGTATATGTCGGTATGTCTTCTTGTTTTAATTTTCCA 981 ATGGCGGAGAACCCTAATTGCACTGTATATGTCGGTATGTCTTCTTGTTTTAATTTTCCA F5 ATGGCGGAGAACCCTAATTGCACTGTATATGTCGGTATGTCTTCTTGTTTTAATTTTCCA F7 ATGGCGGAGAACCCTAATTGCACTGTATATGTCGGTATGTCTTCTTGTTTTAATTTTCCA Favi-9 ATGGCGGAGAACCCTAATTGCACTGTATATGTCGGTATGTCTTCTTGTTTTAATTTTCCA Solyc11g039950.1 ATGGCGGAGAACCCTAATTGCACTGTATATGTCGGTATGTCTTCTTGTTTTAATTTTCCA

967 TATATCTACTTTAATAATTTTTTTATGAACGTTTCTCGTATATCAGATCTGATGTGAAGG 981 TATATCTACTTTAATAATTTTTTTATGAACGTTTCTCGTATATCAGATCTGATGTGAAGG F5 TATATCTACTTTAATAATTTTTTTATGAACGTTTCTCGTATATCAGATCTGATGTGAAGG F7 TATATCTACTTTAATAATTTTTTTATGAACGTTTCTCGTATATCAGATCTGATGTGAAGG Favi-9 TATATCTACTTTAATAATTTTTTTATGAACGTTTCTCGTATATCAGATCTGATGTGAAGG Solyc11g039950.1 TATATCTACTTTAATAATTTTTTTATGAACGTTTCTCGTATATCAGATCTGATGTGAAGG

967 AAGCAATAATCTTAGAGATACGTGCCTTTTCTGGTTATGTTTTCCGATTTGTTTGGTTGA 981 AAGCAATAATCTTAGAGATACGTGCCTTTTCTGGTTATGTTTTCCGATTTGTTTGGTTGA F5 AAGCAATAATCTTAGAGATACGTGCCTTTTCTGGTTATGTTTTCCGATTTGTTTGGTTGA F7 AAGCAATAATCTTAGAGATACGTGCCTTTTCTGGTTATGTTTTCCGATTTGTTTGGTTGA Favi-9 AAGCAATAATCTTAGAGATACGTGCCTTTTCTGGTTATGTTTTCCGATTTGTTTGGTTGA Solyc11g039950.1 AAGCAATAATCTTAGAGATACGTGCCTTTTCTGGTTATGTTTTCCGATTTGTTTGGTTGA

967 TTGATTACTGAATTTATTTAATTTGTTTTTCCCCTTTCTAAACACAGTTGAAAACAACTC 981 TTGATTACTGAATTTATTTAATTTGTTTTTCCCCTTTCTAAACACAGTTGAAAACAACTC F5 TTGATTACTGAATTTATTTAATTTGTTTTTCCCCTTTCTAAACACAGTTGAAAACAACTC F7 TTGATTACTGAATTTATTTAATTTGTTTTTCCCCTTTCTAAACACAGTTGAAAACAACTC Favi-9 TTGATTACTGAATTTATTTAATTTGTTTTTCCCCTTTCTAAACACAGTTGAAAACAACTC Solyc11g039950.1 TTGATTACTGAATTTATTTAATTTGTTTTTCCCCTTTCTAAACACAGTTGAAAACAACTC

967 CGCCTTTGGTTTGGTTCCGTTATGCTCCCTTTTTCCTAATCACTTTCCTTTTAGTCTCTT 981 CGCCTTTGGTTTGGTTCCGTTATGCTCCCTTTTTCCTAATCACTTTCCTTTTAGTCTCTT F5 CGCCTTTGGTTTGGTTCCGTTATGCTCCCTTTTTCCTAATCACTTTCCTTTTAGTCTCTT F7 CGCCTTTGGTTTGGTTCCGTTATGCTCCCTTTTTCCTAATCACTTTCCTTTTAGTCTCTT Favi-9 CGCCTTTGGTTTGGTTCCGTTATGCTCCCTTTTTCCTAATCACTTTCCTTTTAGTCTCTT Solyc11g039950.1 CGCCTTTGGTTTGGTTCCGTTATGCTCCCTTTTTCCTAATCACTTTCCTTTTAGTCTCTT

967 TAGTATTCTAAGATGTCTATTTTAATAAGTCACACAAACATATATATGACTAATTTGAGA 981 TAGTATTCTAAGATGTCTATTTTAATAAGTCACACAAACATATATATGACTAATTTGAGA F5 TAGTATTCTAAGATGTCTATTTTAATAAGTCACACAAACATATATATGACTAATTTGAGA F7 TAGTATTCTAAGATGTCTATTTTAATAAGTCACACAAACATATATATGACTAATTTGAGA Favi-9 TAGTATTCTAAGATGTCTATTTTAATAAGTCACACAAACATATATATGACTAATTTGAGA Solyc11g039950.1 TAGTATTCTAAGATGTCTATTTTAATAAGTCACACAAACATATATATGACTAATTTGAGA

967 TGCACTTTTACTCCATATTATGTCAAATAACATCGACCTCCTAGACTACTTCATAATGAC 981 TGCACTTTTACTCCATATTATGTCAAATAACATCGACCTCCTAGACTACTTCATAATGAC F5 TGCACTTTTACTCCATATTATGTCAAATAACATCGACCTCCTAGACTACTTCATAATGAC F7 TGCACTTTTACTCCATATTATGTCAAATAACATCGACCTCCTAGACTACTTCATAATGAC Favi-9 TGCACTTTTACTCCATATTATGTCAAATAACATCGACCCCCTAGACTACTTCATAATGAC Solyc11g039950.1 TGCACTTTTACTCCATATTATGTCAAATAACATCGACCTCCTAGACTACTTCATAATGAC

967 AACCAATATGTACCTTGGGATGAATTGCACATACATTTGGTTAATCTGCCTTTTTTTTGG 981 AACCAATATGTACCTTGGGATGAATTGCACATACATTTGGTTAATCTGCCTTTTTTTTGG F5 AACCAATATGTACCTTGGGATGAATTGCACATACATTTGGTTAATCTGCCTTTTTTTTGG F7 AACCAATATGTACCTTGGGATGAATTGCACATACATTTGGTTAATCTGCCTTTTTTTTGG Favi-9 AACCAATATGTACCTTGGGATGAATTGCACATACATTTGGTTAATCTGCCTTTTTTTTGG Solyc11g039950.1 AACCAATATGTACCTTGGGATGAATTGCACATACATTTGGTTAATCTGCCTTTTTTTTGG

967 ATAGCTCACTTTGTTGAGGCTATTTTCATTTCCTGTTGGTTGTTTGAATTCAATGCAAGA 981 ATAGCTCACTTTGTTGAGGCTATTTTCATTTCCTGTTGGTTGTTTGAATTCAATGCAAGA

Appendix

F5 ATAGCTCACTTTGTTGAGGCTATTTTCATTTCCTGTTGGTTGTTTGAATTCAATGCAAGA F7 ATAGCTCACTTTGTTGAGGCTATTTTCATTTCCTGTTGGTTGTTTGAATTCAATGCAAGA Favi-9 ATAGCTCACTTTGTTGAGGCTATTTTCATTTCCTGTTGGTTGTTTGAATTCAATGCAAGA Solyc11g039950.1 ATAGCTCACTTTGTTGAGGCTATTTTCATTTCCTGTTGGTTGTTTGAATTCAATGCAAGA

967 ATGCGCTTAGCAGGTTGTGCCTTTTTGATAGATAGGACATGCAATTTCTGTTGTGCTGTG 981 ATGCGCTTAGCAGGTTGTGCCTTTTTGATAGATAGGACATGCAATTTCTGTTGTGCTGTG F5 ATGCGCTTAGCAGGTTGTGCCTTTTTGATAGATAGGACATGCAATTTCTGTTGTGCTGTG F7 ATGCGCTTAGCAGGTTGTGCCTTTTTGATAGATAGGACATGCAATTTCTGTTGTGCTGTG Favi-9 ATGCGCTTAGCAGGTTGTGCCTTTTTGATAGATAGGACATGCAATTTCTGTTGTGCTGTG Solyc11g039950.1 ATGCGCTTAGCAGGTTGTGCCTTTTTGATAGATAGGACATGCAATTTCTGTTGTGCTGTG

967 AATGAGTTGTATGATGTACCACCTTCTAACATGTGTTTGAATTTCTTTTCTTTTTTTTTC 981 AATGAGTTGTATGATGTACCACCTTCTAACATGTGTTTGAATTTCTTTTCTTTTTTTTTC F5 AATGAGTTGTATGATGTACCACCTTCTAACATGTGTTTGAATTTCTTTTCTTTTTTTTTC F7 AATGAGTTGTATGATGTACCACCTTCTAACATGTGTTTGAATTTCTTTTCTTTTTTTTTC Favi-9 AATGAGTTGTATGATGTACCACCTTCTAACATGTGTTTGAATTTCTTTTCTTTTTTTTTC Solyc11g039950.1 AATGAGTTGTATGATGTACCACCTTCTAACATGTGTTTGAATTTCTTTTCTTTTTTTTTC

967 GGGTAGGAAATTTGGATGAGAGAGTATGTGATCGTGTACTGTATGACATTCTAATTCAAG 981 GGGTAGGAAATTTGGATGAGAGAGTATGTGATCGTGTACTGTATGACATTCTAATTCAAG F5 GGGTAGGAAATTTGGATGAGAGAGTATGTGATCGTGTACTGTATGACATTCTAATTCAAG F7 GGGTAGGAAATTTGGATGAGAGAGTATGTGATCGTGTACTGTATGACATTCTAATTCAAG Favi-9 GGGTAGGAAATTTGGATGAGAGAGTATGTGATCGTGTACTGTATGACATTCTAATTCAAG Solyc11g039950.1 GGGTAGGAAATTTGGATGAGAGAGTATGTGATCGTGTACTGTATGACATTCTAATTCAAG

967 CAGGGCGTGTTGTTGACTTGTACATTCCTCGTGACAAGGAAACTGATAAGCCTAAAGGCT 981 CAGGGCGTGTTGTTGACTTGTACATTCCTCGTGACAAGGAAACTGATAAGCCTAAAGGTT F5 CAGGGCGTGCTGTTGACTTGTACATTCCTCGTGACAAGGAAACTGATAAGCCTAAAGGTT F7 CAGGGCGTGTTGTTGACTTGTACATTCCTCGTGACAAGGAAACTGATAAGCCTAAAGGTT Favi-9 CAGGGCGTGTTGTTGACTTGTACATTCCTCGTGACAAGGAAACTGATAAGCCTAAAGGTT Solyc11g039950.1 CAGGGCGTGTTGTTGACTTGTACATTCCTCGTGACAAGGAAACTGATAAGCCTAAAGGTT

967 TTGCCTTTACAAAATATGAGACAGAAGAGATTGCAGACTATGCTGTGAAGCTTTTCTCTG 981 TTGCCTTTGCAAAATATGAGACAGAAGAGATTGCAGACTATGCTGTGAAGCTTTTCTCTG F5 TTGCCTTTGCAAAACATGAGACAGAAGAGATTGCAGACTATGCTGTGAAGCTTTTCTCTG F7 TTGCCTTTGCAAAATATGAGACAGAAGAGATTGCAGACTATGCTGTGAAGCTTTTCTCTG Favi-9 TTGCCTTTGCAAAATATGAGACAGAAGAGATTGCAGACTATGCTGTGAAGCTTTTCTCTG Solyc11g039950.1 TTGCCTTTGCAAAATATGAGACAGAAGAGATTGCAGACTATGCTGTGAAGCTTTTCTCTG

967 GTTTAGTGACACTCTACAATAGAACATTAAAATTTGCA 981 GTTTAGTGACACTCTACAATAGAACATTAAAATTTGCA F5 GTTTAGTGACACTCTACAATAGAACATTAAAATTTGCA F7 GTTTAGTGACACTCTACAATAGAACATTAAAATTTGCA Favi-9 GTTTAGTGACACTCTACAATAGAACATTAAAATTTGCA Solyc11g039950.1 GTTTAGTGACACTCTACAATAGAACATTAAAATTTGCA

Appendix

- Nucleotide alignment the genomic DNA amplified PCR product (1,375- 1,815) of the candidate gene Receptor-like kinase from the resistant lines (GF13 X 981 (F7), GF13 X 967 (F5) and Favi-9), susceptible lines (981, 967) compared with the genomic DNA sequence of Receptor-like kinase (Solyc11g011880.1).

1 10 20 30 40 50 60 | | | | | | | 967 ---CTGTTGATGCAGCGTGTTTTATGAGGTACTCGGATAGAGCTTTTTTTGCTGATAACA 981 GGGCTGTTGATGCAGCGTGTTTTATGAGGTACTCGGATAGAGCTTTTTTTGCTGATAACA F5 GGGCTGTTGATGCAGCGTGTTTTATGAGGTACTCGGATAGAGCTTTTTTTGCTGATAACA F7 GGGCTGTTGATGCAGCGTGTTTTATGAGGTACTCGGATAGAGCTTTTTTTGCTGATAACA Favi-9 GGGCTGTTGATGCAGCGTGTTTTATGAGGTACTCGGATAGAGCTTTTTTTGCTGATAACA Solyc11g011880.1 GGGCTGTTGATGCAGCGTGTTTTATGAGGTACTCGGATAGAGCTTTTTTTGCTGATAACA

967 CGACAACTGATATCACACCATTTCTTGGCGGAGGAGGTAGGAAATGAACCTCTCTTTTTC 981 CGACAACTGATATCACACCATTTCTTGGCGGAGGAGGTAGGAAATGAACCTCTCTTTTTC F5 CGACAACTGATATCACACCATTTCTTGGCGGAGGAGGTAGGAAATGAACCTCTCTTTTTC F7 CGACAACTGATATCACACCATTTCTTGGCGGAGGAGGTAGGAAATGAACCTCTCTTTTTC Favi-9 CGACAACTGATATCACACCATTTCTTGGCGGAGGAGGTAGGAAATGAACCTCTCTTTTTC Solyc11g011880.1 CGACAACTGATATCACACCATTTCTTGGCGGAGGAGGTAGGAAATGAACCTCTCTTTTTC

967 TCAACTCTTACTAGTATTAGCTCGTTTTAAGACAAGTTTATGATTGGTGGTGTTGCTTAT 981 TCAACTCTTACTAGTATTAGCTCGTTTTAAGACAAGTTTATGATTGGTGGTGTTGCTTAT F5 TCAACTCTTACTAGTATTAGCTCGTTTTAAGACAAGTTTATGATTGGTGGTGTTGCTTAT F7 TCAACTCTTACTAGTATTAGCTCGTTTTAAGACAAGTTTATGATTGGTGGTGTTGCTTAT Favi-9 TCAACTCTTACTAGTATTAGCTCGTTTTAAGACAAGTTTATGATTGGTGGTGTTGCTTAT Solyc11g011880.1 TCAACTCTTACTAGTATTAGCTCGTTTTAAGACAAGTTTATGATTGGTGGTGTTGCTTAT

967 GCAGGAAGTTCAAACAAGAAGAAAGCCGTCATTATTGGAGGTGTCGTTGGAGGTGTAGGA 981 GCAGGAAGTTCAAACAAGAAGAAAGCCGTCATTATTGGAGGTGTCGTTGGAGGTGTAGGA F5 GCAGGAAGTTCAAACAAGAAGAAAGCCGTCATTATTGGAGGTGTCGTTGGAGGTGTAGGA F7 GCAGGAAGTTCAAACAAGAAGAAAGCCGTCATTATTGGAGGTGTCGTTGGAGGTGTAGGA Favi-9 GCAGGAAGTTCAAACAAGAAGAAAGCCGTCATTATTGGAGGTGTCGTTGGAGGTGTAGGA Solyc11g011880.1 GCAGGAAGTTCAAACAAGAAGAAAGCCGTCATTATTGGAGGTGTCGTTGGAGGTGTAGGA

967 CTTCTTTTGATTGTATTGGCTGTTTTCCTATGGTATCGACTATCAAGAAAGCCAAAGACA 981 CTTCTTTTGATTGTATTGGCTGTTTTCCTATGGTATCGACTATCAAGAAAGCCAAAGACA F5 CTTCTTTTGATTGTATTGGCTGTTTTCCTATGGTATCGACTATCAAGAAAGCCAAAGACA F7 CTTCTTTTGATTGTATTGGCTGTTTTCCTATGGTATCGACTATCAAGAAAGCCAAAGACA Favi-9 CTTCTTTTGATTGTATTGGCTGTTTTCCTATGGTATCGACTATCAAGAAAGCCAAAGACA Solyc11g011880.1 CTTCTTTTGATTGTATTGGCTGTTTTCCTATGGTATCGACTATCAAGAAAGCCAAAGACA

967 GCTGAGAGAGGTCAAATTCGGATGATCATTTCTGAATCTTCTTTAGTTCTGTTTCGTCCT 981 GCTGAGAGAGGTCAAATTCGGATGATCATTTCTGAATCTTCTTTAGTTCTGTTTCGTCCT F5 GCTGAGAGAGGTCAAATTCGGATGATCATTTCTGAATCTTCTTTAGTTCTGTTTCGTCCT F7 GCTGAGAGAGGTCAAATTCGGATGATCATTTCTGAATCTTCTTTAGTTCTGTTTCGTCCT Favi-9 GCTGAGAGAGGTCAAATTCGGATGATCATTTCTGAATCTTCTTTAGTTCTGTTTCGTCCT Solyc11g011880.1 GCTGAGAGAGGTCAAATTCGGATGATCATTTCTGAATCTTCTTTAGTTCTGTTTCGTCCT

967 TCCATTTATTCATGTTGTTAAATGATATAGTAGTTCATATTCAGGTAATATACTGGGAGC 981 TCCATTTATTCATGTTGTTAAATGATATAGTAGTTCATATTCAGGTAATATACTGGGAGC F5 TCCATTTATTCATGTTGTTAAATGATATAGTAGTTCATATTCAGGTAATATACTGGGAGC F7 TCCATTTATTCATGTTGTTAAATGATATAGTAGTTCATATTCAGGTAATATACTGGGAGC Favi-9 TCCATTTATTCATGTTGTTAAATGATATAGTAGTTCATATTCAGGTAATATACTGGGAGC Solyc11g011880.1 TCCATTTATTCATGTTGTTAAATGATATAGTAGTTCATATTCAGGTAATATACTGGGAGC

967 AACTGAGCTGAGAGGCCC--- 981 AACTGAGCTGAGAGGCCCGGT F5 AACTGAGCTGAGAGGCCCGGT F7 AACTGAGCTGAGAGGCCCGGT Favi-9 AACTGAGCTGAGAGGCCCGGT Solyc11g011880.1 AACTGAGCTGAGAGGCCCGGT

Appendix

- Nucleotide alignment the genomic DNA amplified PCR product (2,726- 3,147) of the candidate gene Receptor-like kinase from the resistant lines (GF13 X 981 (F7), GF13 X 967 (F5) and Favi-9), susceptible lines (981, 967) compared with the genomic DNA sequence of Receptor-like kinase (Solyc11g011880.1).

1 10 20 30 40 50 60 | | | | | | | 967 TCAGTGGAAGGAGGAGCAACGATATGCAAATCGAGCCTGTCACTGAATATCTACTCGAAC 981 TCAGTGGAAGGAGGAGCAACGATATGCAAATCGAGCCTGTCACTGAATATCTACTCGAAC F5 TCAGTGGAAGGAGGAGCAACGATATGCAAATCGAGCCTGTCACTGAATATCTACTCGAAC F7 TCAGTGGAAGGAGGAGCAACGATATGCAAATCGAGCCTGTCACTGAATATCTACTCGAAC Favi-9 TCAGTGGAAGGAGGAGCAACGATATGCAAATCGAGCCTGTCACTGAATATCTACTCGAAC Solyc11g011880.1 TCAGTGGAAGGAGGAGCAACGATATGCAAATCGAGCCTGTCACTGAATATCTACTCGAAC

967 AGGTCTGTTCTCATTTTCAAATATACAATCTTTGTTTGAGTTAATGATAAAACACATCTA 981 AGGTCTGTTCTCATTTTCAAATATACAATCTTTGTTTGAGTTAATGATAAAACACATCTA F5 AGGTCTGTTCTCATTTTCAAATATACAATCTTTCTTTGAGTTAATGATAAAACACATCTA F7 AGGTCTGTTCTCATTTTCAAATATACAATCTTTCTTTGAGTTAATGATAAAACACATCTA Favi-9 AGGTCTGTTCTCATTTTCAAATATACAATCTTTGTTTGAGTTAATGATAAAACACATCTA Solyc11g011880.1 AGGTCTGTTCTCATTTTCAAATATACAATCTTTGTTTGAGTTAATGATAAAACACATCTA

967 AACTATCACCTTTTCGCGAGTCCTATCTAATCTTGGCATGGTGAAATAGGCGTGGAAGCT 981 AACTATCACCTTTTCGCGAGTCCTATCTAATCTTGGCATGGTGAAATAGGCGTGGAAGCT F5 AACTATCACCTTTTCGCGAGTCCTATCTAATCTTGGCATGGTGAAATAGGCGTGGAAGCT F7 AACTATCACCTTTTCGCGAGTCCTATCTAATCTTGGCATGGTGAAATAGGCGTGGAAGCT Favi-9 AACTATCACCTTTTCGCGAGTCCTATCTAATCTTGGCATGGTGAAATAGGCGTGGAAGCT Solyc11g011880.1 AACTATCACCTTTTCGCGAGTCCTATCTAATCTTGGCATGGTGAAATAGGCGTGGAAGCT

967 TCATGAAACTGGCACGCCTGTAAAACTAGTGGACGAGACATTAGACCCCAACGAATACAA 981 TCATGAAACTGGCACGCCTGTAAAACTAGTGGACGAGACATTAGACCCCAACGAATACAA F5 TCATGAAACTGGCACGCCTGTAAAACTAGTGGACGAGACATTAGACCCCAACGAATACAA F7 TCATGAAACTGGCACGCCTGTAAAACTAGTGGACGAGACATTAGACCCCAACGAATACAA Favi-9 TCATGAAACTGGCACGCCTGTAAAACTAGTGGACGAGACATTAGACCCCAACGAATACAA Solyc11g011880.1 TCATGAAACTGGCACGCCTGTAAAACTAGTGGACGAGACATTAGACCCCAACGAATACAA

967 CGAACAAGAAGTGAAGAAAGTCATAGAGATCGCGTTAATGTGCACACAGTCACCAGCAAA 981 CGAACAAGAAGTGAAGAAAGTCATAGAGATCGCGTTAATGTGCACACAGTCACCAGCAAA F5 CGAACAAGAAGTGAAGAAAGTCATAGAGATCGCGTTAATGTGCACACAGTCACCAGCAAA F7 CGAACAAGAAGTGAAGAAAGTCATAGAGATCGCGTTAATGTGCACACAGTCACCAGCAAA Favi-9 CGAACAAGAAGTGAAGAAAGTCATAGAGATCGCGTTAATGTGCACACAGTCACCAGCAAA Solyc11g011880.1 CGAACAAGAAGTGAAGAAAGTCATAGAGATCGCGTTAATGTGCACACAGTCACCAGCAAA

967 TCTTAGGCCAAGCATGTCTGAAGTTGTTGTGATGCTATTAAGCGATCGTAGCACAGAATC 981 TCTTAGGCCAAGCATGTCTGAAGTTGTTGTGATGCTATTAAGCGATCGTAGCACAGAATC F5 TCTTAGGCCAAGCATGTCTGAAGTTGTTGTGATGCTATTAAGCGATCGTAGCACAGAATC F7 TCTTAGGCCAAGCATGTCTGAAGTTGTTGTGATGCTATTAAGCGATCGTAGCACAGAATC Favi-9 TCTTAGGCCAAGCATGTCTGAAGTTGTTGTGATGCTATTAAGCGATCGTAGCACAGAATC Solyc11g011880.1 TCTTAGGCCAAGCATGTCTGAAGTTGTTGTGATGCTATTAAGCGATCGTAGCACAGAATC

967 AAGAACTCCAAGCAGGCCTACTATCATTAGCATGGATAAGAGTAAAGCATTCGACGCATC 981 AAGAACTCCAAGCAGGCCTACTATCATTAGCATGGATAAGAGTAAAGCATTCGACGCATC F5 AAGAACTCCAAGCAGGCCTACTATCATTAGCATGGATAAGAGTAAAGCATTCGACGCATC F7 AAGAACTCCAAGCAGGCCTACTATCATTAGCATGGATAAGAGTAAAGCATTCGACGCATC Favi-9 AAGAACTCCAAGCAGGCCTACTATCATTAGCATGGATAAGAGTAAAGCATTCGACGCATC Solyc11g011880.1 AAGAACTCCAAGCAGGCCTACTATCATTAGCATGGATAAGAGTAAAGCATTCGACGCATC

967 CA 981 CA F5 CA F7 CA Favi-9 CA Solyc11g011880.1 CA

Appendix

- Nucleotide alignment the genomic DNA amplified PCR product (1-800) of the candidate gene LRR-RLP from the resistant lines (GF13 X 981 (F7), GF13 X 967 (F5) and Favi-9), susceptible lines (981, 967) compared with the genomic DNA sequence of LRR-RLP (Solyc11g016930.1).

967 CTTCTAGATTATTGTCAAAGGTGTGTACTGTACAACTTGAGTATACTTTTTTTTTGTTAC 981 CTTCTAGATTATTGTCAAAGGTGTGTACTGTACAACTTGAGTATACTTTTTTTTTGTTAC F5 CTTCTAGATTATTGTCAAAGGTGTGTATTGTACTACTTGAGTATACTTTTTTTTTGTTAC F7 CTTCTAGATTATTGTCAAAGGTGTGTATTGTACTACTTGAGTATACTTTTTTTTTGTTAC Favi-9 CTTCTAGATTATTGTCAAAGGTGTGTATTGTACTACTTGAGTATACTTTTTTTTTGTTAC Solyc11g016930.1 CTTCTAGATTATTGTCAAAGGTGTGTACTGTACAACTTGAGTATACTTTTTTTTTGTTAC

967 TATTCTATGTTGTGAAGGTCTTTTAGAAGCATGTATTTTGTACTACTAAAATAGAGTATA 981 TATTCTATGTTGTGAAGGTCTTTTAGAAGCATGTATTTTGTACTACTAAAATAGAGTATA F5 TATTCTATGTTGTGAAGGTCTTTTAGAAGCATGTATATTGTACTACTAAAATAGAGTATA F7 TATTCTATGTTGTGAAGGTCTTTTAGAAGCATGTATATTGTACTACTAAAATAGAGTATA Favi-9 TATTCTATGTTGTGAAGGTCTTTTAGAAGCATGTATATTGTACTACTAAAATAGAGTATA Solyc11g016930.1 TATTCTATGTTGTGAAGGTCTTTTAGAAGCATGTATTTTGTACTACTAAAATAGAGTATA

967 TTTGATGGGAAAGTTGTGCATACAAATCTGCAAGTAATATATGATAGTTAAACTATTTCT 981 TTTGATGGGAAAGTTGTGCATACAAATCTGCAAGTAATATATGATAGTTAAACTATTTCT F5 TTTGATGGGAAAGTTGTGCATACAAATCTGCAAGTAATATATGATAGTTAACCTATTTGT F7 TTTGATGGGAAAGTTGTGCATACAAATCTGCAAGTAATATATGATAGTTAACCTATTTGT Favi-9 TTTGATGGGAAAGTTGTGCATACAAATCTGCAAGTAATATATGATAGTTAACCTATTTGT Solyc11g016930.1 TTTGATGGGAAAGTTGTGCATACAAATCTGCAAGTAATATATGATAGTTAAACTATTTCT

967 TAACAGTTCAGTTTATTTATATGACCGCACTAAAACCAAGTAGCAAAACTGTGTTTTCCT 981 TAACAGTTCAGTTTATTTATATGACCGCACTAAAACCAAGTAGCAAAACTGTGTTTTCCT F5 TAACAGTTCAGTTTATTTATATGACCGCACTAAAACCAAGTAGCAAAACTGTGTTTTCCT F7 TAACAGTTCAGTTTATTTATATGACCGCACTAAAACCAAGTAGCAAAACTGTGTTTTCCT Favi-9 TAACAGTTCAGTTTATTTATATGACCGCACTAAAACCAAGTAGCAAAACTGTGTTTTCCT Solyc11g016930.1 TAACAGTTCAGTTTATTTATATGACCGCACTAAAACCAAGTAGCAAAACTGTGTTTTCCT

967 CTGTCTGTTTCTCTGTGTAGGACTGTAAGTATATTAGTTAGAATTTATTTAATTGATAGT 981 CTGTCTGTTTCTCTGTGTAGGACTGTAAGTATATTAGTTAGAATTTATTTAATTGATAGT F5 CTGTCTGTTTCTCTGTGTAGGACTGTAAGTATATTAGTTAAAATTTATTTAATTGATAGT F7 CTGTCTGTTTCTCTGTGTAGGACTGTAAGTATATTAGTTAAAATTTATTTAATTGATAGT Favi-9 CTGTCTGTTTCTCTGTGTAGGACTGTAAGTATATTAGTTAAAATTTATTTAATTGATAGT Solyc11g016930.1 CTGTCTGTTTCTCTGTGTAGGACTGTAAGTATATTAGTTAGAATTTATTTAATTGATAGT

967 TGAATTATTAAATATAAGTATCTGTTTTGGGTTTGTGTTAATAGTTTA-TTAATCATTCA 981 TGAATTATTAAATATAAGTATCTGTTTTGGGTTTGTGTTAATAGTTTA-TTAATCATTCA F5 TGAATTATTAAATATAAGTATCTGTTTTGGGTTTGTGTTAATAGTTTAATTAATCATTCA F7 TGAATTATTAAATATAAGTATCTGTTTTGGGTTTGTGTTAATAGTTTAATTAATCATTCA Favi-9 TGAATTATTAAATATAAGTATCTGTTTTGGGTTTGTGTTAATAGTTTAATTAATCATTCA Solyc11g016930.1 TGAATTATTAAATATAAGTATCTGTTTTGGGTTTGTGTTAATAGTTTA-TTAATCATTCA

967 TTTGTAAGAAATGAGTGATGAAGAAAAATCCCTGATGCATCTTCCATCTCCTATCTTGTT 981 TTTGTAAGAAATGAGTGATGAAGAAAAATCCCTGATGCATCTTCCATCTCCTATCTTGTT F5 TTTGTAAGAAATGAGTGATGAAGAAAAATCCCTGATGCATCTTCCATCTCCTATCTTGTT F7 TTTGTAAGAAATGAGTGATGAAGAAAAATCCCTGATGCATCTTCCATCTCCTATCTTGTT Favi-9 TTTGTAAGAAATGAGTGATGAAGAAAAATCCCTGATGCATCTTCCATCTCCTATCTTGTT Solyc11g016930.1 TTTGTAAGAAATGAGTGATGAAGAAAAATCCCTGATGCATCTTCCATCTCCTATCTTGTT

967 ACAAATTCCCTCTACATTACCTCGTACCACACTTTTACACGTCAAAACCCTCTCCAAATC 981 ACAAATTCCCTCTACATTACCTCGTACCACACTTTTACACGTCAAAACCCTCTCCAAATC F5 ACAAATTCCCTCTACATTACCTCGTACCACACTCTTACACGTCAAAACCCTCTCCAAATC

Appendix

F7 ACAAATTCCCTCTACATTACCTCGTACCACACTCTTACACGTCAAAACCCTCTCCAAATC Favi-9 ACAAATTCCCTCTACATTACCTCGTACCACACTCTTACACGTCAAAACCCTCTCCAAATC Solyc11g016930.1 ACAAATTCCCTCTACATTACCTCGTACCACACTTTTACACGTCAAAACCCTCTCCAAATC

967 TTATCTAAATCTAACCTTAAATTCCGAATTTTTAATAATGTCACGTTCAGCATCTCCAGC 981 TTATCTAAATCTAACCTTAAATTCCGAATTTTTAATAATGTCACGTTCAGCATCTCCAGC F5 TTATCTAAATCTAACCTTAAATTCCGAATTTTTAATAATGTCACGTTCAGCATCTCCAGC F7 TTATCTAAATCTAACCTTAAATTCCGAATTTTTAATAATGTCACGTTCAGCATCTCCAGC Favi-9 TTATCTAAATCTAACCTTAAATTCCGAATTTTTAATAATGTCACGTTCAGCATCTCCAGC Solyc11g016930.1 TTATCTAAATCTAACCTTAAATTCCGAATTTTTAATAATGTCACGTTCAGCATCTCCAGC

967 AAGCATCATCAGCCAATTCAACTCTTTTTGGATTAACAGCTTAAAATTGTTGAGATTCGT 981 AAGCATCATCAGCCAATTCAACTCTTTTTGGATTAACAGCTTAAAATTGTTGAGATTCGT F5 AAGCATCATCAGCCAATTCAACTCTTTTTGGATTAACAGCTTAAAATTGTTGAGATTCGT F7 AAGCATCATCAGCCAATTCAACTCTTTTTGGATTAACAGCTTAAAATTGTTGAGATTCGT Favi-9 AAGCATCATCAGCCAATTCAACTCTTTTTGGATTAACAGCTTAAAATTGTTGAGATTCGT Solyc11g016930.1 AAGCATCATCAGCCAATTCAACTCTTTTTGGATTAACAGCTTAAAATTGTTGAGATTCGT

967 TTGTGTCGATAATAACTGTGATCATGATCCACATGTTGACCTTCATCTGAGACTTTCCTT 981 TTGTGTCGATAATAACTGTGATCATGATCCACATGTTGACCTTCATCTGAGACTTTCCTT F5 TTGTGTCGATAATAACTGTGATCATGATCCACATGTTGACCTTCATCTGAGACTTTCCTT F7 TTGTGTCGATAATAACTGTGATCATGATCCACATGTTGACCTTCATCTGAGACCTTCCTT Favi-9 TTGTGTCGATAATAACTGTGATCATGATCCACATGTTGACCTTCATCTGAGACTTTCCTT Solyc11g016930.1 TTGTGTCGATAATAACTGTGATCATGATCCACATGTTGACCTTCATCTGAGACTTTCCTT

967 TCCAATAGATCCATTTTTCCTCGTTGGATCGGTTCATGGTTTTGTTTGTTTTAATAGCTT 981 TCCAATAGATCCATTTTTCCTCGTTGGATCGGTTCATGGTTTTGTTTGTTTTAATAGCTT F5 TCCAATAGATCCATTTTTCCTCGTTGGATCGGTTCATGGTTTTGTTTGTTTTAATAGCTT F7 TCCAATAGATCCATTTTTCCTCGCTGGATCGGTTCATGGTTTTGTTTGTTTTAATAGCTT Favi-9 TCCAATAGATCCATTTTTCCTCGTTGGATCGGTTCATGGTTTTGTTTGTTTTAATAGCTT Solyc11g016930.1 TCCAATAGATCCATTTTTCCTCGTTGGATCGGTTCATGGTTTTGTTTGTTTTAATAGCTT

967 TGTCGGTGATGCTGATAGTAT 981 TGTCGGTGATGCTGATAGTAT F5 TGTCGGTGATGCTGATAGTAT F7 TGTCGGTGATGCTGATAGTAT Favi-9 TGTCGGTGATGCTGATAGTAT Solyc11g016930.1 TGTCGGTGATGCTGATAGTAT

Appendix

- Nucleotide alignment the genomic DNA amplified PCR product (3,239- 4,046) of the candidate gene LRR-RLP from the resistant lines (GF13 X 981 (F7), GF13 X 967 (F5) and Favi-9), susceptible lines (981, 967) compared with the genomic DNA sequence of LRR-RLP (Solyc11g016930.1).

1 10 20 30 40 50 60 | | | | | | | 967 GAAGGAGAGGTTGTGAATGTCTTTTAGATTGTCAAAATGTATGTACTGTTTCATTTTAGT 981 GAAGGAGAGGTTGTGAATGTCTTTTAGATTGTCAAAATGTATGTACTGTTTCATTTTAGT F5 GAAGGAGAGGTTGTGAATGTCTTTTAGATTGTCAAAATGTATGTACTGTTTCATTTTAGT F7 GAAGGAGAGGTTGTGAATGTCTTTTAGATTGTCAAAATGTATGTACTGTTTCATTTTAGT Favi-9 GAAGGAGAGGTTGTGAATGTCTTTTAGATTGTCAAAATGTATGTACTGTTTCATTTTAGT Solyc11g016930.1 GAAGGAGAGGTTGTGAATGTCTTTTAGATTGTCAAAATGTATGTACTGTTTCATTTTAGT

967 GACTGTGATGTGAGTTCTTGCTTTGGGTTAAAGACTTTGTATTCTGACTTTGCATAATAC 981 GACTGTGATGTGAGTTCTTGCTTTGGGTTAAAGACTTTGTATTCTGACTTTGCATAATAC F5 GACTGGGATGTGAGTTCTTGCTTTGGGTTAAAGACTTTGTATTCTGACTTTGCATAATAC F7 GACTGTGATGTGAGTTCTTGCTTTGGGTTAAAGACTTTGTATTCTGACTTTGCATAATAC Favi-9 GACTGTGATGTGAGTTCTTGCTTTGGGTTAAAGACTTTGTATTCTGACTTTGCATAATAC Solyc11g016930.1 GACTGTGATGTGAGTTCTTGCTTTGGGTTAAAGACTTTGTATTCTGACTTTGCATAATAC

967 ATAAAACGTGTTCTTTAACTTGGCTTCGTAAGGAATTATGTTCTTCAATTTTGGATGGAT 981 ATAAAACGTGTTCTTTAACTTGGCTTTGTAAGGAATTATGTTCTTCAATTTTGGATGGAT F5 ATAAAACGTGTTCTTTAACTTGGCTTCGTAAGGAATTATGTTCTTCAATTTTGGATGGAT F7 ATAAAACGTGTTCTTTAACTTGGCTTCGTAAGGAATTATGTTCTTCAATTTTGGATGGAT Favi-9 ATAAAACGTGTTCTTTAACTTGGCTTCGTAAGGAATTATGTTCTTCAATTTTGGATGGAT Solyc11g016930.1 ATAAAACGTGTTCTTTAACTTGGCTTCGTAAGGAATTATGTTCTTCAATTTTGGATGGAT

967 AACAAATAGACACAACTCTATCTACGATATTTTTTCTTTCATTTTCAGTGCTACTTCACA 981 AACAAATAGACACAACTCTACCTACAATATTTTTTCTTTCATTTTCAGTGCTACTTCACA F5 AACAAATAGACACAACTCTATCTACAATATTTTTTCTTTCATTTTCAGTGCTACTTCACA F7 AACAAATAGACACAACTCTATCTACAACATTTTTTCTTTCATTTTCAGTGCTACTTCACA Favi-9 AACAAATAGACACAACTCTATCTACAATATTTTTTCTTTCATTTTCAGTGCTACTTCACA Solyc11g016930.1 AACAAATAGACACAACTCTATCTACAATATTTTTTCTTTCATTTTCAGTGCTACTTCACA

967 ATGTTACTGCATCAAATGATATTAAGTGCTTGCAAGGACTGAAGGACTCATTTAAGGATC 981 ATGTTACTGCATCAAATGATATTAAGTGCTTGCAAGGACTGAAGGACTCATTTAAGGATC F5 ATGTTACTGCATCAAATGATATTAAGTGCTTGCAAGGACTGAAGGACTCATTTAAGGATC F7 ATGTTACTGCATCAAATGATATTAAGTGCTTGCAAGGACTGAAGGACTCATTTAAGGATC Favi-9 ATGTTACTGCATCAAATGATATTAAGTGCTTGCAAGGACTGAAGGACCCATTTAAGGATC Solyc11g016930.1 ATGTTACTGCATCAAATGATATTAAGTGCTTGCAAGGACTGAAGGACTCATTTAAGGATC

967 CTAATGTAAATTTCAATTCTTGGAACTTCTCAAACTACTCCATGGGGTTTATCTGCAAGT 981 CTAATGTAAATTTCAATTCTTGGAACTTCTCAAACTACTCCATGGGGTTTATCTGCAAGT F5 CTAATGTAAATTTCAATTCTTGGAACTTCTCAAACTACTCCATGGGGTTTATCTGCAAGT F7 CTAATGTAAATTTCAATTCTTGGAACTTCTCAAACTACTCCATGGGGTTTATCTGCAAGT Favi-9 CTAATGTAAATTTCAATTCTTGGAACTTCTCAAACTACTCCATGGGGTTTATCTGCAAGT Solyc11g016930.1 CTAATGTAAATTTCAATTCTTGGAACTTCTCAAACTACTCCATGGGGTTTATCTGCAAGT

967 TTGTTGGTGTCATTTACTGGAACAACCTCGAGAACCGGATGATCAGCCTCTCACTTCCAA 981 TTGTTGGTGTCATTTACTGGAACAACCTCGAGAACCGGATGATCAGCCTCTCACTTCCAA F5 TTGTTGGTGTCATTTACTGGAACAACCTCGAGAACCGGATGATCAGCCTCTCACTTCCAA F7 TTGTTGGTGTCATTTACTGGAACAACCTCGAGAACCGGATGATCAGCCTCTCACTTCCAA Favi-9 TTGTTGGTGTCATTTACTGGAACAACCTCGAGAACCGGATGATCAGCCTCTCACTTCCAA Solyc11g016930.1 TTGTTGGTGTCATTTACTGGAACAACCTCGAGAACCGGATGATCAGCCTCTCACTTCCAA

967 ACATGAATCTCAGTGTACAGCTCCCAGATGCCTTCAAATGTTGCTCATCTTTGGCTACTC 981 ACATGAATCTCAGTGTACAGCTCCCAGATGCCTTCAAATGTTGCTCATCTTTGGCTACTC F5 ACATGAATCTCAGTGTACAGCTCCCAGATGCCTTCAAATGTTGCTCATCTTTGGCTACTC F7 ACATGAATCTCAGTGTACAGCTCCCAGATGCCTTCAAATGTTGCTCATCTTTGGCTACTC Favi-9 ACATGAATCTCAGTGTACAGCTCCCAGATGCCTTCAAATGTTGCTCATCTTTGGCTACTC Solyc11g016930.1 ACATGAATCTCAGTGTACAGCTCCCAGATGCCTTCAAATGTTGCTCATCTTTGGCTACTC

967 TTCATCTCTCTGGTAACAGGTTCTCCGGTCCCATTCCTTCAGAGATTTGTAGTTGGACTG 981 TTCATCTCTCTGGTAACAGGTTCTCCGGTCCCATTCCTTCAGAGATTTGTAGTTGGACTG F5 TTCATCTCTCTGGTAACAGGTTCTGCGGTCCCATTCCTTCAGAGATTTGTAGTTGGACTG

Appendix

F7 TTCATCTCTCTGGTAACAGGTTCTGCGGTCCCATTCCTTCAGAGATTTGTAGTTGGACTG Favi-9 TTCATCTCTCTGGTAACAGGTTCTGCGGTCCCATTCCTTCAGAGATTTGTAGTTGGACTG Solyc11g016930.1 TTCATCTCTCTGGTAACAGGTTCTCCGGTCCCATTCCTTCAGAGATTTGTAGTTGGACTG

967 CATATTTAGTAAAACTCGACTTGT------CAGTTTCTCTGGCTCTATACCTGCTGAAC 981 CATATTTAGTAAAACTCGACTTGT------CAGTTTCTCTGGCTCTATACCTGCTGAAC F5 CATATTTAGTAAAACTCGACTTGTCTAACAACAGTTTCTCTGGCTCTATACCTGCTGAAC F7 CATATTTAGTAAAACTCGACTTGTCTAACAACAGTTTCTCTGGCTCTATACCTGCTGAAC Favi-9 CATATTTAGTAAAACTCGACTTGTCTAACAACAGTTTCTCTGGCTCTATACCTGCTGAAC Solyc11g016930.1 CATATTTAGTAAAACTCGACTTGT------CAGTTTCTCTGGCTCTATACCTGCTGAAC

967 TTAGAAACTGCACCTACTTGAACAAGTTGATTCTCAATAACAACAAACTATCTGGAAATA 981 TTAGAAACTGCACCTACTTGAACAAGTTGATTCTCAATAACAACAAACTATCTGGAAATA F5 TTGGAAACTGCACCTACTTGAACAAGTTGATTCTCAATAACAACAAACTATCTGGAAATA F7 TTGGAAACTGCACCTACTTGAACAAGTTGATTCTCAATAACAACAAACTATCTGGAAATA Favi-9 TTGGAAACTGCACCTACTTGAACAAGTTGATTCTCAATAACAACAAACTATCTGGAAATA Solyc11g016930.1 TTAGAAACTGCACCTACTTGAACAAGTTGATTCTCAATAACAACAAACTATCTGGAAATA

967 TTCCACCAGAAATCTCTCAATTAACAAGGCTCAAGGTGCTCTCCTTGGCTAACAACAATC 981 TTCCACCAGAAATCTCTCAATTAACAAGGCTCAAGGTGCTCTCCTTGGCTAACAACAATC F5 TTCCACCAGAAATCTCTCAATTAACAAGGCTCAAGGTGCTCTCCTTGGCTAACAACAATC F7 TTCCACCAGAAATCTCTCAATTAACAAGGCTCAAGGTGCTCTCCTTGGCTAACAACAATC Favi-9 TTCCACCAGAAATCTCTCAATTAACAAGGCTCAAGGTGCTCTCCTTGGCTAACAACAATC Solyc11g016930.1 TTCCACCAGAAATCTCTCAATTAACAAGGCTCAAGGTGCTCTCCTTGGCTAACAACAATC

967 TTTCTGGTAACATACCACCATTCTCAGGATTGACTGATTTTGAGTATGGAGGAAATAGAC 981 TTTCTGGTAACATACCACCATTCTCAGGATTGACTGATTTTGAGTATGGAGGAAATAGAC F5 TTTCTGGTAACATACCACCGTTCTCAGGATTGACTGATTTTGAGTATGGAGGAAATAGAC F7 TTTCTGGTAACATACCACCATTCTCAGGATTGACTGATTTTGAGTATGGAGGAAATAGAC Favi-9 TTTCTGGTAACATACCACCATTCTCAGGATTGACTGATTTTGAGTATGGAGGAAATAGAC Solyc11g016930.1 TTTCTGGTAACATACCACCATTCTCAGGATTGACTGATTTTGAGTATGGAGGAAATAGAC

967 ATCTCTGCGGTGGAACATTAGCCAAATGTGGTTGA 981 ATCTCTGCGGTGGAACATTAGCCAAATGTGGTTGA F5 ATCTCTGCGGTGGAACATTAGCCAAATGTGGTTGA F7 ATCTCTGCGGTGGAACATTAGCCAAATGTGGTTGA Favi-9 ATCTCTGCGGTGGAACATTAGCCAAATGTGGTTGA Solyc11g016930.1 ATCTCTGCGGTGGAACATTAGCCAAATGTGGTTGA

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Appendix

- Nucleotide sequence of TYLCV isolate

1 10 20 30 40 50 60 70 | | | | | | | | ACCGGATGGCCGCGCCTTTTTCTTTTATGTGGTCCCCACGAGGGTTCCACAGACGTCACTGTCAACCAAT CAAATTGCATCCTCAAACGTTAGATAAGTGTTTATTTGTCTTTATATACTTGGTCCCCAAGTTTTTTGTC TTGCAATATGTGGGACCCACTTCTAAATGAATTTCCTGAATCTGTTCACGGATTTCGTTGTATGTTAGCT ATTAAATATTTGCAGTCCGTTGAGGAAACTTACGAGCCCAATACATTGGGCCACGATTTAATTAGGGATC TTATATCTGTTGTAAGGGCCCGTGACTATGTCGAAGCGACCAGGCGATATAATCATTTCCACGCCCGTCT CGAAGGTTCGCCGAAGGCTGAACTTCGACAGCCCATACAGCAGCCGTGCTGCTGTCCCCATTGTCCAAGG CACAAACAAGCGACGATCATGGACGTACAGGCCCATGTACCGAAAGCCCAGAATATACAGAATGTATCGA AGCCCTGATGTTCCCCGTGGATGTGAAGGCCCATGTAAAGTCCAATCTTTTGAGCAACGGGATGATATTA AGCATACTGGTATTGTTCGTTGTGTTAGTGATGTTACTCGTGGATCTGGAATTACTCACAGAGTGGGTAA GAGGTTCTGTGTTAAATCGATATATTTTTTAGGTAAAGTCTGGATGGATGAAAATATCAAGAAGCAGAAT

CACACTAATCAGGTCATGTTCTTCTTGGTCCGTGATAGAAGGCCCTATGGAAACAGTCCAATGGATTTTG GACAGGTTTTTAATATGTTCGATAATGAGCCCAGTACCGCAACCGTGAAGAATGATTTGCGGGATAGGTT TCAAGTGATGAGGAAATTTCATGCTACAGTTATTGGTGGGCCTTCTGGAATGAAGGAACAGGCATTAGTT AAGAGATTTTTTAAAATTAACAGTCATGTAACTTATAATCATCAGGAGGCAGCCAAGTACGAGAACCATA CTGAAAACGCCTTGTTATTGTATATGGCATGTACGCATGCCTCTAATCCAGTGTATGCAACTATGAAAAT ACGCATCTATTTCTATGATTCAATATCAAATTAATAAAATTTATATTTTATATCATGAGTTTCGGTTACA TTTATTGTGTTTTCAACTACATCATACAATACATGATCAACTGCTCTGATTACATTGTTAATGGAAATTA CACCAAGACTATCTAAATACTTAATAACTTCATATCTAAATACTCTTAAGAAACGACCAGTCTGAGGCTG TAATGTCGTCCAAATTCGGAAGTTGAGAAAACATTTGTGAATCCCCATTACCTTCCTGATGTTGTGGTTG AATCTTATCTGAATGGAAATGATGTCGTGGTTCATTAGAAATGGCCTGTGGCTGTGTTCTGTTATCTTGA

AATATAGGGGATTGTTTATCTCCCAGATAAAAACGCCATTCTCTGCCTGAGGAGCAGTGATGAGTTCCCC TGTGCGTGAATCCATGATTACTGCAGTTGAGGTGGAGGTAGTATGAGCAGCCACAGTCTAGGTCTACACG CTTACGCCTTATTGGTTTCTTCTTGGCTATCTTGTGTTGGACCTTGATTGATACTTGCGAACAGTGGCTC GTAGAGGGTGACGAAGGTTGCATTCTTGAGAGCCCAATTTTTCAAGGAAAAGTTTTTTTCTTCGTCTAGA TATTCCCTATATGAGGAGGTAGGTCCTGGATTGCAGAGGAAGATAGTGGGAATTCCCCCTTTAATTTGAA TGGGCTTCCCGTACTTTGTGTTGCTTTGCCAGTCCCTCTGGGCCCCCATGAATTCCTTGAAGTGCTTTAA ATAATGCGGGTCTACGTCATCAATGACGTTGTACCACGCATCATTACTGTACACCTTTGGGCTTAGGTCT AGATGTCCACATAAATAATTATGTGGGCCTAGAGACCTGGCCCACATTGTTTTGCCTGTTCTGCTATCAC CCTCAATGATAATACTATAAAGGTCTCCATGGCCGCGCAACCGAGACACGACGTTCTCGGACACCCATAC TTCAAGTTCATCTGGAACTTGATTAAAAGATGAAGATAAAAAGGGAGAAATATAAGGAGCCGGAGGCTCC

TGAAAAATTCTATCTAAATTACTATTTAAATTATGAAACTGTAAAATATAATCTTTCGGTACTAATTCTT TAATGATTCTAAGAGCCTCTGACTTACTGCCTGAGTTAAGAGCTGCGGCGTAAGCGTCATTGGCTGACTG CTGACCCCCACGTGCAGATCGTCCGTCGATCTGAAACTCACCCCAGTCGACGGTGTCTCCGTCCTTATCG ACATAAGACTTGACGTCTGAACTGGATTTAGCTCCCTGAATGTTTGGATGGAAATGTGCTGCCCTGCTTG GGGATACCAGGTCGAAGAATCGCTGATTTTGGCATTTGAATTTCCCTTCAAATTGGATAAGCACATGGAG ATGTGGTTCCCCATTCTCGTGGAGTTCTCTGCAAACTTTGATGTATTTTTTATTTGTTGGGGTTTCTAGG TTTTTTAATTGGGAAAGTGCTTCTTCTTTTGTTAAGGAGCAATGAGGATATGTGAGGAAATAATTTTTGC AATTTATTTGGAAGCGCTTAGGAGGAGCCATATGGTCAATGAGTACCGATTGACCAAGATTTTTACACTT ATCCCTGGTGTATCGGTACTCAATATATAGTGAGTACCAAATGGCATTTTGGTAATAACATAAAAGTACA TTGCAATTCAAAATTCAAAATTAAAAAATCAAATCATTAAAGCGGCCATCCGTATAATATT

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Acknowledgement

8. Acknowledgement

Thanking God is the first and the most important part of this acknowledgement. God always gives me patience and strong desire to complete this part of my life.

There are many people without whom this thesis might not have been completed. I wish I could express my appreciation to them.

To finish this work. My supervisor Prof. Dr. Uwe Sonnewald offered a lot of help, patience, and guidance. For him I would like to say Thank you very much.

Many thanks are extended to many people in the chair of Biochemistry who helped me directly and indirectly during my work. So many thanks to Stephen Reid, Sophia Sonnewald, Jörg Hofmann, Bushra Amin, Tissue culture and greenhouse team, Ingrid Schießl. I would like to thank my colleagues and friend: Marlene, Marlies, Eva, Haina, Jessica, Kathrin, and Susanna who shared me many memories during my stay in Germany.

Many thanks and a lot of appreciation to Prof. Dr. Ghandi Anfoka (Al- Balqaa Applies University) who always supporting me and lit my way to scientific research.

My precious father, I would like to thank you millions of times for encouraging me to achieve what I am looking for. The unconditional love and frequent advices from you and my beloved mother were the source of my inspiration.

Special Thanks to my beloved husband Mohammad, he was supporting me as much as he could. My son Talal also supported me as he was a motive force for me to complete this hard job.

It is of great value to thank my beloved brothers and sisters (Riyad, Husam, Eman, Amal, Osama, Somaia, Roqaia, and Rajaa), their spouses and kids, and many close relatives who always surrounded me with warm feelings and hope.

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Acknowledgement

I would like to thank my marvelous friends: Hanadi Ghanem, Marwa Azzam, Yasmin Hussain, and Inas Shaaban for their help and support.

Even though their names haven’t been mentioned, many thanks to my friends who were caring, supporting, and made my life more colorful.

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