Resistance (R) Genes; a Natural Resource of Plant-Pathogen Resistance

DEPARTMENT OF BIOLOGICAL AND ENVIRONMENTAL SCIENCES

Resistance (R) genes; a natural resource of plant-pathogen resistance

Iman Abbas

Degree project for Bachelor of Science with a major in Biology BIO602 Biology; Degree project 15 Hec Second cycle Semester/year: Autumn 2018 Supervisor: Mats Andersson, Department of Biological and Environmental Science Examiner: Cornelia Spetea-Wiklund, Department of Biological and Environmental Science

TABLE OF CONTENTS

Abstract ...... 2

Sammanfattning ...... 3

Forewords ...... 4

Introduction ...... 5 Plant immunity ...... 5 R genes and R proteins ...... 6 Aim ...... 7

Material and methods ...... 8 Study design ...... 8 Datamining and article selections ...... 8 Article review ...... 9

Results ...... 9 Recognition mechanisms ...... 10 Direct and indirect interactions ...... 11 Evolution and diversifying mechanisms of R genes ...... 11 Breeding for more resistant plants ...... 13

Discussion and conclusions ...... 16

Acknowledgments ...... 19

References ...... 20

Appendix 1, Search Schedule ...... 22

ABSTRACT Breeding for more disease-resistant plants has been an important subject to secure global food production. Focus has been on breeding of crops such as maize, rice, and wheat, which are consumed widely and form the basis of staple foods for many people. This literature study discusses the possibility to control and improve plant disease resistance by the use of resistance (R) genes in modern crop-breeding programs. PubMed Central provides scientific articles for this literature study. The majority of R proteins belong to the NB-LRR family consisting of two domains, NB-ARC and LRR. Both these domains play a central role in the activation of plant- pathogen recognition inducing a defense response in the plant cell. The dynamics of plant- pathogen interaction suggests a great variation and diversity of R genes in the plant kingdom. Protein Data Bank provides 3D structures of NB-ARC Apfa-1 (1Z6T), the LRR homodimer Lr10 (4V2D), TIR and CC domains of the L6 (3OZI) and Mla10 (3QFL). The organization of R proteins is well preserved throughout generations in coevolved populations. They are therefore considered to be a great natural resource of resistance to control and improve plant disease resistance. The use of a single R gene-mediated resistance in traditional crop-breeding programs has its limitations and is therefore not durable as a long-term solution. The conclusion that a single R gene-mediated resistance is not durable shed light on the inherited quantitative traits of these genes. Combining the quantitative traits of these genes in a sustainable way with other development strategies in customized crop-breeding programs may provide a long-term solution. The involvement of multiple R gene-mediated resistance has shown great potential as an environmentally friendly way to improve and control plant disease resistance.

Keywords: Plant immunity, R genes, NB-LRR, Breeding program, Development strategies.

SAMMANFATTNING För att säkra den globala livsmedelsproduktionen har förädling av mer sjukdomsresistenta växter blivit ett viktigt ämne. Fokus har varit på grödor som konsumeras globalt och utgör basfödan för många människor, såsom majs, ris och vete. Denna litteraturstudie kommer därför att diskutera möjligheten att använda resistans (R) gener i moderna förädlings-program med syfte att kontrollera samt förbättra motståndet hos växter gentemot patogener. PubMed Central har försett denna litteraturstudie med vetenskapliga artiklar. Majoriteten av R proteiner tillhör familjen NB- LRR vilket består av två huvuddomäner, NB-ARC och LRR. Båda spelar en central roll i aktiveringen av växt-patogenigenkänningen som inducerar växtcellensförvar. Dynamiken i växt- patogeninteraktionerna antyder på att det finns en stor variation och mångfald av R gener i växtriket. Protein Data Bank har försett denna litteraturstudie med tredimensionella strukturer av NB-ARC Apfa-1 (1Z6T), LRR homodimer Lr10 (4V2D), TIR- och CC-domänen av L6 (3OZI) och Mla10 (3QFL). Strukturen hos R proteiner är väl bevarade inom den genetiska polen i samevolutionära populationer. Därför anses de vara en stor naturlig resurs för kontroll och förbättring av växternas resistens. Användning av traditionella förädlings-program som medför R-genmedierat försvar bestående av en singel R-gen har visats ha sina begränsningar och leder därför till kortsiktig resistens gentemot patogener. Slutsatsen att en singel R-genmedierat försvar inte är hållbart uppmärksammade R-geners kvantitativa egenskaper. Egenskaperna i en multipel R-genmedierat försvar kan kombineras med andra förädlingsutvecklingsstrategier för att uppnå ett hållbart samt långsiktig resistens gentemot växtsjukdomar. En multipel R-genmedierat försvar har därför visats ha stor potential som ett miljövänligt sätt för att kontrollera samt förbättra växtresistensen.

Nyckelord: Växtimmunitet, R-gener, NBS-LRR, Förädlingsprogram, Utvecklingsstrategier.

FOREWORDS

“Go back young man and gather up your weary and defeated genes of the past, take your currently successful genes, find some new ones if you can, and build yourself a highly durable strategy.”

Richard R. Nelson

INTRODUCTION

PLANT IMMUNITY Plants have developed a two-layered immune system to defend themselves against pathogens in the agricultural ecosystems. The two-layered plant immunity contains several disease resistance components. The first-layer includes physical barriers and pattern recognition receptors (PRRs). A defense response triggered by the activation of PRRs is led by a system known as the pattern- triggered immunity. The second-layer includes proteins encoded by R genes, which recognize specific pathogen effectors. A defense response triggered by R proteins results in a strong and rapid response leading to a localized effector-triggered immunity. The recognition induces the synthesis of antimicrobial enzymes and other metabolites that generate downstream signaling. These pathways activate the defense in neighboring cells surrounding the site of the infection and often lead to programmed cell death (Dangl et al. 2013). The need for matched specificity between the R protein and pathogen avirulence effector (Avr) was first presented by Flor’s gene-for-gene hypothesis. Flor describe correlation between the inherited resistance and pathogenicity in linseed rust caused by fungus (Melampsora lini). He described the correlation with “for each gene conditioning rust reaction in the host there is a specific gene conditioning pathogenicity in the parasite” (Flor, 1971). The concept has been applied to various degree of proof to other host-pathogen interactions including bacteria, viruses, fungi and nematodes (Sasaki, 2000). Different schools of thought later expanded the gene-for- gene hypothesis. According to Vanderplank (Vanderplank, 1978) the specificity in gene-for-gene lies in the susceptibility. Later on, he elaborated the protein-for-protein hypothesis as a biochemical explanation of the gene-for-gene hypothesis. The biochemical explanation states that in gene-for-gene diseases the mutual recognition of host and pathogen is not by their genes themselves but by their encoded proteins. Vanderplank hypothesized that in susceptibility the pathogen injects an Avr effector into the host cell, that interacts with a complementary R protein triggering a defense response. The induced defense response in plant-pathogen recognition is described by Stackman’s hypersensitivity (HR). Stackman proposed year 1915 HR as a term that described the rapid defense response leading to programmed cell death, collapsing the tissue surrounding the infection area to restrict and prevent any further infection development (Mur et al. 2008). These hypotheses have been of great importance for the understanding of plant disease resistance and an excellent contribution to plant pathology. Plant disease resistance has a global interest due to its crucial impact on food production. The reduction of quality foods impacts human health and economic creating a dependence on imported foods. A reduction of quality food could also mean that communities have to replace a balanced diet with processed foods which could create further health problems. The interest to understand these R genes and their functions awoken during the 90s leading to cloning of the first R gene. Hm1 from maize (Zea mays) was the first cloned R gene that provides resistance against the fungus Cochliobolus carbonum. Since then several R genes have been cloned such as N from tobacco, Pto and Cf-9 from tomato (Kourelis & Van der Hoorn 2018). One of the most inexpensive and environmentally friendly ways to control plant diseases is to identify resistant variants that target specific pathogenic strains. Incorporating this knowledge into new crop-breeding programs could result in more disease resistant plants (Dangl et al. 2013).

R GENES AND R PROTEINS The plant kingdom has thousands of R genes, whose proteins mediate resistance against specific pathogens such as fungal, bacterial or viral strains. The recognition is facilitated either by direct or indirect interactions. There is no clear suggestion on how large the proportion of all plant- pathogen interactions are direct or indirect, there is however an indication that the indirect interaction predominates (Kourelis & Van der Hoorn 2018). The direct plant-pathogen interaction (R1, Figure 1) describes the mechanism where a pathogen contains an Avr effector and injects it into the plant cell. The recognition will occur through the binding affinity of an R protein and Avr effector inducing a defense response (Dangl et al. 2013). The indirect plant-pathogen interaction is described by the guard hypothesis, where the Avr effectors are injected into the plant cell to target specific protein functions. R proteins function as “guards” that detect a change of normal activity in the plant cell inducing a defense response (Guard, Figure 1, Van der Hoorn & Kamoun 2008). The majority of R proteins belong to the NB-LRR family. NB-LRR proteins consist of two central domains, a nucleotide-binding (NB-ARC) and a leucine-rich repeat (LRR) domain. The NB-LRR proteins can also be further divided into two receptor groups; Toll/interleukin-1 receptors (TIR) and coiled-coil receptors (CC) (Figure 2, Takken & Goverse 2012).

Figure 1. Plant-pathogen interactions. The direct interaction (R1): Avr effector binds through its affinity to the corresponding R protein inducing a defense response. The indirect interaction (Guard): Avr effector is introduced into the plant cell targeting specific protein functions. The corresponding R protein detects the change in plant activity inducing a defense response (Figure prepared based on findings in Dangl et al. 2013, Van der Hoorn & Kamoun 2008).

Figure 2. TIR and CC receptors. The domains are shown in different colors; TIR (white): Toll/interleukin-1 receptor; CC (white): Coiled-coil receptor; NB-ARC (light grey) and LRR (dark grey) (Figure prepared based on findings in Takken & Goverse 2012).

The NB-ARC domain occurs in various proteins with ATP or GTP binding activity, suggesting that it executes the function of a molecular switch. The activation of these R proteins is controlled by the binding of ATP. The ATP-bound state alters the interaction between R proteins and other members of the signal transduction cascade. The LRR domain occurs in various proteins of the innate immunity providing recognition of Avr effectors. The LRR domain also facilitates the interaction between R proteins and other members of the signal transduction chain (Figure 3, Takken & Goverse 2012).

Figure 3. The different domains of NB-LRR proteins. The 3D structures of the NB-ARC are based on the human structure Apaf-1 (PDB ID 1Z6T). The LRR is based on the homodimer structure Lr10 (PDB ID 4V2D). The TIR/CC domains are of the L6 (PDB ID 3OZI) and M1a10 (PDB ID 3QFL, Protein Data Bank (https://www.rcsb.org), modified from figure 1 in Takken & Goverse 2012).

AIM The evolving plant pathogens in the agricultural ecosystems have been a subject of interests in a global matter due to their crucial importance on both human health and economics as well as environmentally. This literature study will therefore discuss the possibility to improve resistance in plants by the use of R genes.

The following questions will be addressed: - Why are R genes considered to be a natural resource of resistance in plants? - Is R gene-mediated resistance durable as a long-term solution?

MATERIAL AND METHODS

STUDY DESIGN The following study is designed as a literature study. Literature studies are made by systematically searching and comparing scientific articles to provide an overview of a specific area (Zainal, 2007).

DATAMINING AND ARTICLE SELECTIONS To ensure there was enough material to complete this literature study, an initial search was conducted. The initial search was made to identify keywords relevant to the study. These keywords were then used in the actual search that was made in PubMed Central (https://www.ncbi.nlm.nih.gov/pmc/), which is the United State National Library of Medicine providing digital archives of scientific journal literature. PubMed Central is an open-access database where the search for full articles is done by either writing author name, topic or title and search across all articles. PubMed Central can also be accessed through NCBI (https://www.ncbi.nlm.nih.gov). The search for articles was then divided into research categories such as the evolutionary development and diversity of the R gene in plants, the protein structure and functions of NB-LRR genes, mechanism of the plant-pathogen interactions as well as breeding development strategies. The search was conducted in English and thus keywords that were used were “NB-LRR”, “plant- pathogen interactions”, “plant resistance”, “R genes and plant-breeding”. Keywords were then used and combined in different ways, see appendix 1. The selection of articles was made using the Boolean search operator AND to see which relationship the different keywords had to each other. The operator AND was also used to assemble search terms as well as to limit the search. In the actual search truncation and phrase search where also used. Truncation was used to expand the search, by searching for different word endings or different variants of a word. Phrase search limits the search results and provides relevant articles to a specific topic. The actual search resulted in 29 relevant articles, 22 of them were genome analysis studies and 7 of them were review articles. Manual searches were also made using the reference list in relevant articles and resulted in 6 additional articles leading to a total of 35 articles (Figure 4).

PubMed Central

Initial search

Actual search Keywords (5111)

Relevant articles Rejected articles (29) (5082)

Manual search (6)

Figure 4. Representation of the article search. An initial and actual search conducted in PubMed Central identifying keywords and scientific articles. Actual search: 5111; 29 relevant, 5082 rejected, manual search; 6 articles.

An additional search was made in Protein Data Bank (https://www.rcsb.org), which is a freely accessible database providing 3D structures of biological proteins based on various search options. The search was made using the PDB ID of the human structure of NB-ARC Apfa-1 (1Z6T), the LRR homodimer Lr10 (4V2D), TIR and CC domains of the L6 (3OZI) and Mla10 (3QFL). The search resulted in 3D structures of each domain as well as information such as release date, authors name and protein classification. The selected 3D structures were then downloaded as a picture and used in the introduction section.

ARTICLE REVIEW The article reviews began with reading the titles of the articles in each category. Based on the relevance of the headlines, the abstract was then read to get an overview of the article. Articles that did not serve the purpose of the literature study were then excluded. The articles were read several times, separately and then jointly to compare the content of the articles.

RESULTS The structure and function of each domain of R proteins have now been discussed, but how are the genes coding for these proteins organized in the plant genome? Table 1 shows a large range of identified R genes among the different plant species. The R genes were identified within the different plant genomes in preserved clusters. Porter et al. 9

(2009) identified 54 R genes in papaya (Carica papaya), while Shang et al. (2009) identified 653 R genes in rice (Oryza sativa L. spp. indica).

Table 1. Identified R genes in various plant species. Plant species Identified TIR CC Reference R genes

Papaya 54 7 6 Porter et al. (2009) (Carica papaya) Saffron crocus 57 13 18 Wan et al. (2013) (Cucumis sativus) Field mustard 92 62 30 Mun et al. (2009) (Brassica rapa) Stiff brome 126 0 113 Tan S & Wu S (2012) (Brachypodium distachyon) Thale cress 149 94 55 Meyers et al. (2003) (Arabidopsis thaliana) Birdsfoot trefoil 158 32 28 Li et al. (2010) (Lotus japonicus) Sand cress 185 123 38 Guo et al. (2011) (Arabidopsis lyrata) Soybean 319 - - Kang et al. (2012) (Glycine max) Barrel clover 333 156 177 Kohler et al. (2008) (Medicago truncatula) Black cottonwood 402 91 119 Guo et al. (2008) (Populus trichocarpa) Potato 438 77 361 Lozano et al. (2012) (Solanum tuberosum) Grape wine 459 97 203 Yang et al. (2008) (Vitis vinifera) Japanese rice 553 - - Shang et al. (2009) (Orzya sativa L. spp. Japonica) Indian rice 653 - - Shang et al. (2009) (Orzya sativa L. spp. Indica) The identified R genes in various plant species can be further divided into TIR and CC receptors depending on their encoded R proteins.

RECOGNITION MECHANISMS The need for matched specificity between R proteins and their corresponding Avr effectors presented by Flor’s and Vanderplank’s hypothesis does not indicate that there is only one R gene/R protein for a given Avr gene/Avr effector. The recognition can 10

therefore be mediated by several mechanisms (table 2). Mathieu et al. (2014) show how one Avr effector is recognized by two R proteins. Meyers et al. (2003) show on the other hand how two different Avr effectors are recognized by the same R protein. Lida et al. (2015) show a third type of pathogen recognition mechanism where two R genes essentially encode identical R proteins resulting in the recognition of the same Avr effector.

Table 2. Several R-Avr recognitions. R protein Plant Avr effector Pathogen Reference

Pto Tomato avrPto P.s. pv. tomato Mathieu et al. (2014)

Prf Tomato avrPto P.s. tomato Mathieu et al. (2014) avrPtoB RPM1 Arabidopsis avrRpm1 P.s. pv. maculicola Meyers et al. (2003) avrB Cf-9 Tomato avr9/avr2 C. fulvum Lida et al. (2015)

Cf-2 Tomato avr2/avr9 C. fulvum Lida et al. (2015)

DIRECT AND INDIRECT INTERACTIONS The simplest recognition of matched specificity is through direct interaction. Dodds et al. (2006) show the direct interaction mechanism in flax. This plant species has 12 allelic variants of the L locus, which encode L proteins that interact directly with the corresponding AvrL567 from the flax rust fungus Melampsora lini. This study provided evidence that the L protein was responsible for the recognition of the pathogen. A yeast two-hybrid experiment indicated that the resistance proteins L5, L6 and L7 bind to specific variants of AvrL567 effectors. Dodds et al. (2006) further observed the interaction of the inactive form of the RRS1 protein with the corresponding Avr PopP2 in a yeast two-hybrid experiment. The Prf protein shows the indirect interaction in tomato. Prf is involved in the indirect detection of two effectors, AvrPto and AvrPtoB found in Pseudomonas syringae. The study by Mathieu et al. 2014 indicates that Prf is necessary for the activation of the defense response.

EVOLUTION AND DIVERSIFYING MECHANISMS OF R GENES Since Darwin’s natural selection theory in the Origin of Species, coevolution was recognized as a fundamental driver of evolution in interacting species. The dynamics of plant-pathogen interactions have been described in two models, the Red Queen hypothesis and the Arms race hypothesis. The Red Queen hypothesis is described with “running as fast as you can to stay in the same place”, to prevent extinction. It proposes the idea that accumulating adaptions in one species increases the fitness against another species in tight coevolved interactions. Such adaptation change would also cause a decline in the fitness of the second species, which could lead to extinction (Figure 5a). The second evolutionary hypothesis, Arms race, describes coevolved adaptions in both populations through a slow process (Figure 5b, Brockhurst et al. 2014).

Figure 5. The evolutionary theories describing the law of excitations in coevolved species. Red Queen hypothesis: describes the adaption of two coevolved species (grey host, black pathogen) in a population as a result of allele frequency and through generations. Arms Race hypothesis: describes the adaption of two species (grey host, black pathogen) in populations as a result of allele frequency sweeps through generations (Modified from figure 1 in Lighten et al. 2017).

Plant-pathogen interactions described using the Red Queen hypothesis demonstrates how genetic variation within the R genes is preserved in a population throughout generations. Jensen et al. (2012) present in a study the resistance differences in two barley populations that clearly endorse the Red Queen hypothesis for local adaption. According to the Red Queen hypothesis, a pathogen and plants have two alleles: Avr1 and Avr2, and R1 and R2, respectively. When a pathogen infects a plant by introducing Avr1 into its population, the frequency selection will respond with the production of the corresponding R1 allele. R1 will dominate the population with a high frequency resulting in the reduction of Avr1 affectivity. This selection will benefit the Avr2 allele, introducing it into the plant population. As a result, the plant population will increase the frequency of the corresponding R2 allele, which will on the other hand decrease the R1. The plant population is now locally adaptable to the most common allele Avr2 and not resistant against Avr1. As a result of the local adaption, the pathogen will benefit from the original allele Avr1. The pathogen will be able to infect the plant population with the low-frequency allele Avr1 and start a new cycle. Results presented by Jensen et al. (2012) show the frequency selection in two barley populations. The first barley population responded with an increased resistance due to the continuous competition of evolving pathogens. Meanwhile, the second population of barley showed that the absence of Avr loci in the pathogen population resulted in the disappearance of old R alleles in the plant population. The LRR region of NB-LRRs has been shown to be a subject for positive selection. Meyers et al. (2002) present in genome sequence study evidence that LRRs have regions that are subjected to positive selection by analyzing the different ratios of non-synonyms (dN) to

synonyms (dS) SNPs in a gene sequence. A non-synonym SNP is a base substitution in a codon that changes amino acid while a synonym SNP is a base substitution that results in the same amino acid. When dN/dS=1, a gene is evolving naturally which is a result of non-synonymous and synonymous mutations accumulating with the same velocity. In contrast when dN/dS>1, a gene accumulates more non-synonymous SNPs which may indicate positive selection. Meyers et al. (2002) applied the dN/dS to identify positive selection within the LRRs region in 116 R genes from Arabidopsis, revealing significant dN/dS-values (P<0,0001) in 83 of the 116 genes. Polymorphism such as SNPs has been shown to be of great interest. Okuyama et al. (2011) present a study about the rice (Oryza sativa) resistance gene Pia that provides resistance against fungus (Magnaporthe oryzea) carrying the Pi-Avr gene. The sequencing detected a G/T base substitution in the TGA-codon at position +1396, which causes an amino acid change from cytosine to phenylalanine at the NB domain. Similarly, Zhang et al. (2015) presents a study where a G/A substitution at position +1183 was found, which causes an alteration of the donor- splicing site resulting in premature termination of translation at position +1276. Rice carrying the wild-type Pia resistance gene provided resistance against fungus while those carrying the G/T- SNP in the NBSA+1396-locus or G/A-SNP in the NBSA+1183 -locus were more susceptible of the infection. There are also other types of mutations that can affect the velocity in the genetic evolution and provide a brought spectrum of resistance, such as duplications, recombination, unequal crossing over and gene conversions. Gene duplication is an important mechanism to provide new gene functions and to create genetic novelty. Many new functions have evolved through gene duplication and have been recognized as a diversifying force contributing to the genetic evolution. Zhong et al. (2015) demonstrated the specific duplication of NB-LRR genes in five species: strawberry (Fragaria vesca), apple (Malus domestica), pear (Pyrus × bretschneideri), peach (Prunus persica) and mei (Prunus mume). The study shows a high percentage of NB-LRR genes derived by specific duplications. Among the five genomes, apple showed the highest percentage, namely 68% of the NB-LRR genes were derived by duplications. Strawberry had 62%, pear 49%, mei 40% and 37% in peach. This indicates that recent duplications have an essential role in the contribution to the expansion of NB-LRR genes in these five species. Mondragon-Palomino and Guat (2006) discuss the evolutionary impacts of unequal crossing over and gene conversion in the recognition regions of NB-LRR in Arabidopsis thaliana. Unequal crossing over and gene conversion are biological factors that could affect the test statistics when positive selection is tested. Mondragon-Palomino and Guat (2006) discuss therefore the overlap between unequal crossing over/gene conversion and positive selection in the LRR region of the NB-LRR genes in Arabidopsis thaliana. In this study, the data were selected from the database for gene homologs of NBS-LRR disease resistance genes and were detected by the GENECONV 1.18 method. The method tests unequal crossing over/gene conversion by identifying identical nucleotide fragments. The GENECONV 1.18 method was based on cluster- alignment and subfamily-alignment which showed statistically significant levels (P<0,05). The significant levels in the NB-LRR recognition regions indicate that unequal crossing over/gene conversion has been an important force in the expansion and diversity among R genes.

BREEDING FOR MORE RESISTANT PLANTS R genes have been considered as an important natural resource for resistance, but are the R gene- mediated resistance durable? The plant-pathogen interactions, diversity and genetic evolutionary mechanisms have been discussed in the previous parts of this literature study, providing an overview for the understanding of the role of R genes. R proteins will fail to recognize the evolved Avr effectors, which usually results in the introduction of new R genes leading to the boom-and-bust cycle of disease. By definition, the "boom" will occur when a new R gene is introduced into agriculture providing a high degree of resistance and also adapted into larger 13

agricultural landscapes. The "bust" will occur when a pathogen population emerges, which will reduce the resistance efficiency leading to an epidemic (Figure 6, Bruns 2017).

Acreage Frequency increases increases

Cultivar Increased Host with new Host Epidemic Pathogen virulence resistance detection

Acreage Frequency decreases decreases

Figure 6. The boom-and-bust cycle of disease. A cultivar mix with new R genes is prepared and introduced into a population. The introduction of the new cultivar mix results in a high degree of resistance in the population. The corresponding pathogen will emerge with high frequency decreasing the resistance within the population resulting in an epidemic (Figure prepared based on findings in Bruns 2017).

A good example that shows the boom-and-bust cycle is presented in a review article by Herbert Bruns (2017) discussing the 1970’s southern corn (Zea mays) leaf blight (SCLB) epidemics. The origin of the epidemics began with the discovery of the male sterility gene T-urf13 that encodes a suppressor protein, leading to reduced pollen production. This discovery led to the genetic development of new cultivars containing the male fertility gene Rf2 that increased the seed corn production. The hybrid cultivar cms-T was widely adopted in larger areas in the US increasing the production by >85%. However, an unidentified disease was later on discovered to affect the plants. Experimental studies showed that plants from the hybrid cms-T cultivars were highly susceptible to the virulent pathogen, indicating the loss of the R gene. In contrast, cultivars without the cms-T showed no symptoms and were resistant to the fungus (Cochliobolus heterostrophus) infection. The disease outbreak caused serious damage to the corn production with an estimated loss of $1.0 billion. With this in mind, which lessons should we learn today from the SCLB epidemic of the 1970s? A statement by Ullstrup (1972) reads: “never again should a major cultivated species be molded into such uniformity that is so universally vulnerable to attack by a pathogen, an insect, or environmental stress. Diversity must be maintained in both the genetic and cytoplasmic constitution of all-important crop species”. Durable resistance is by definition a long-term effective resistance in a cultivar that is widely grown in an environment favorable to the disease. Resistance can however be divided into horizontal (HR) and vertical resistance (VR). HR describes the involvement of multiple genes providing a high degree of resistance during long-term periods. In contrast, VR describes the involvement of a single gene with the disadvantage of providing resistance during short-term periods. Keane (2012) discuss in a study the advantages and disadvantages of HR and VR in potato blight caused by oomycete (Phytophthora infestans). This experimental study shows how cultivated potato species are susceptible to the fungus infection: meanwhile, wild collected potato species containing other R genes were resistant to the disease. Single R genes were crossbred into the cultivated potato species, resulting in some species being immune to specific isolates of the

fungus but susceptible to others in the pathogen population. This indicates a VR due to the single gene efficiency that during long-term will decrease and break down. Multiple R genes were later on crossbred into the potato cultivars showing high efficiency of resistance. The fundamental differences are that VR provides high resistance to only one race and very low to others. Meanwhile, HR is not race-specific and is therefore considered to be more durable. How will plant breeders break the boom-and-bust cycle and attain durability? A statement presented in the conference, Novel approaches to achieve durable disease resistance (2015) reads: “it is time to consider optimized development strategies for resistance genes in agroecosystems with the aim of achieving sustainable, durable disease resistance”. Brun et al, (2010) show in an experimental analysis the advantages of customized quantitative resistance (QR) strategy. By definition, the QR method is dependent on the involvement of multiple R genes that are associated with quantitative trait loci (QTL). QR is difficult to apply since it is dependent on a normally distributed phenotype providing partial resistance effects due to the involvement of multiple R genes. Brun et al. (2010) designed a long-term experiment using two types of properties (B. napus), one involving the presence or absence of the Lm6 R gene. The other one involving the presence or absence of QR exhibited in high glucosinolate compounds. Lm6 is an R gene that provides resistance against the fungus Leptosphaeria maculans. Glucosinolates are secondary metabolites found in the Brassicaceae family and are active substance in the defense cascade against pathogens. An interesting advantage of this experimental design is the use of natural pathogen inoculation that is expected to occur in agricultural fields. This experiment shows the emerge of Lm6 corresponding Avr gene (after three years) in the population lacking QR, while the population carrying the QR shows a lack of developing the corresponding Avr. It is worth to mention for higher credibility of these results that the experiments were geographically separated by 1000 m. The aim of this separation is to limit the inoculation movement between experiments to prevent cross-contamination. How can the knowledge of the R gene role and QR provide durable resistance? There are many gene development strategies that can be implicated and of use in modern crop-breeding programs such as gene rotation, gene pyramids, cultivar mixtures or a combination of those. Gene rotation is a breeding strategy describing the development of an effective R gene replacement. The strategy is based on the introduction of an alternative R gene replacing the original R gene when the corresponding Avr appear in the population. The disadvantage associated with the gene rotation strategy is the difficulty of monitoring the Avr appearance accurately in agricultural farms, having to prepare cultivars with an effective R gene replacement as well as simultaneously change cultivars throughout larger agricultural landscapes. Even if these requirements were met, the frequency of Avr may also not decrease once the corresponding R gene is removed. The gene pyramids are by definition a combination of several R genes deployed against pathogens to increase the resistance durability. McCallum et al. (2012) present a good example of modern disease control using the Lr13 in combination with other R genes providing a high degree of resistance against wheat leaf rust. Lr13 gene pyramid combination was presented in a cultivar that maintained durable resistance against wheat leaf rust (Puccinia) from the years of 1991-2014 (Figure 7).

Figure 7. Gene pyramid. R gene combination Lr30 and Lr34 provides low levels of resistance to leaf Lr30 rust. R gene combination Lr11, Lr13, Lr34 and Lr16 provide high levels of resistance to leaf rust. R gene Lr34 combination Sr22, Sr33, Sr35, Sr45 and Sr50 provides resistance to stem leaf rust. Combining all of these R genes in a cultivar mixture provides durable resistance (Figure prepared based on Lr11 Lr13 findings in McCallum et al. 2012). Lr34 Lr16

Sr22 Sr33 Sr35 Sr45 Sr50

Various cultivar mixtures can also be combined within an agricultural landscape providing several degrees of resistance against pathogens. Originally, the cultivar mixtures were developed as a two-component mixture containing a susceptible cultivar and one totally resistant cultivar. Sapoukhina et al. (2013) showed decreased levels of wheat yellow rust in cultivar mixtures containing R genes. The three-year field experiment used six single seed descent (SSD) wheat lines crossed with two cultivar mixtures, namely the susceptible Yr6 and the resistant Yr17 gene. This study also discussed enhanced resistance effects by different proportions of resistance cultivar. An effective disease reduction of 45% was shown in the wheat line, by only adding a small amount of the resistant and susceptible mixture (R/S). An additional increase of the R- mixture provided a higher degree of resistance, namely 60% in the wheat lines. An important mentioning of this field experiment is that two lines of SSD wheat were planted and arranged in different blocks with four replications throughout the agricultural field. Each block was geographically separated by 1000 m to prevent cross-contamination of the result.

DISCUSSION AND CONCLUSIONS Since Borlaug’s statement (1970) “civilization as it is known today could not have evolved, nor can it survive, without an adequate food supply" has the breeding for more resistant crops been an important quest to secure global food production. Agrochemicals play an essential role in the reduction losses of crop production. However, these agrochemicals have serious impacts on humans and environment. This literature study has discussed the potential use of R genes as a natural resource of resistance in agricultural systems providing more sustainable and durable resistance against pathogens. The first part of this literature study provides an overview of the R gene and R proteins role in the innate immunity of plants. The second part of this literature study has discussed the possibility to implicate the knowledge of these genes in modern crop-breeding programs with the aim to develop strategies that provide more durable resistance against evolved and newly developed pathogen strains. Why are these genes recognized as a natural resource of resistance? The genomic organization of these genes shows a great variation and diversity of R genes that provide a broad spectrum of resistance. The broad spectrum of resistance can be used and modified in terms to strengthen the crop ability to defend themselves by their natural way against various pathogens. The genomic organization of these genes show also one interesting similarity through the

irregular distribution, the majority of R genes are organized in conserved clusters among the different plant species. Their conserved cluster organization provides a great advantage for these genes by preserving them throughout generations. Furthermore, the evolutionary and diversifying mechanisms suggest that these genes are well preserved throughout generations due to various evolutionary mechanisms and biological factors. The Red Queen and Arms Race hypotheses have shown to be a fundamental driver of tight coevolved populations, preserving R alleles throughout generations even if their efficiency has decreased. In some cases, it appears to be cyclic and the result of frequency depended selections, in other cases, it has shown to be the result of several selective sweeps. In either case, the R alleles are preserved in the population with high or low- frequency levels and can therefore be of use depending on the coevolved adaptions in both populations. Jensen et al. (2012) show also the importance of the continuous competition between the adaptions in coevolved populations that would preserve old alleles within the genetic pole. The experiment shows how old R alleles is preserved within the genetic pole in the first population due to the continuous competition of the evolving pathogens. In the second population, old R alleles disappeared due to the absence of the corresponding Avr loci in the pathogen population. The Red Queen and Arms race hypotheses also indicate that the diversity of R genes could be a reflection of the Avr gene variation in coevolved populations. Dodds et al. (2006) mention an interesting observation that corroborates this suggestion in the yeast two-hybrid experiment of the RRS1 protein and its corresponding Avr PopP2 effector protein. These experiments show how gene-for-gene interactions are a result of a gene-specific arms race that leads to the variation of R and Avr genes. Further evidence suggests that R genes are sustainable and can be of great use as a natural resource for more resistant crops is that they are adaptable and coevolves with pathogens. The gene-for-gene hypothesis corroborates the suggestion on a coevolved development in the plant-pathogen system through the binding affinity. The recognition of the Avr effector proteins indicates that the affinity lies between the specificity between the products of the R and Avr genes. On the other hand, there is one example mentioned in this literature study that shows how an R allele can recognize several Avr effector proteins. Meyers et al. (2003) show how RPM1 can interact with two Avr effectors, avrRmp1 and avrB. This is incompatible with the gene-for-gene hypothesis discussed in this literature study since an R allele that can recognize and bind to several effector proteins suggest that the affinity is not entirely specific. However, the gene-for-gene hypothesis indicates that several R genes can interact and provide resistance of a quantitative trait. One example that provides insight of the advantages of their quantitative trait is presented by Keane (2012), that shows how the resistance efficiency of a single crossbred R gene into a potato species decreases and breaks down in the long-term. On the contrary, when multiple R genes are crossbred into the same potato species providing resistance of a quantitative trait, resulting with high resistance efficiency that did not decrease or break down long-term. The advantage of the quantitative traits of the multiple R genes is that they are not race-specific and are therefore more usable as a long-term solution in terms of unknown and newly developed pathogens in a population. The insight of their quantitative traits of these genes is of importance to the second part of this literature study. The use of R gene-mediated resistance in traditional breeding programs may provide a short- term solution. The resistance will decrease and break down due to the rapid evolving Avr phenotype in the agricultural ecosystems leading to the boom-and-bust cycle of disease. One example discussed in this study is the Southern corn (Z. mays) leaf blight (SCLB) epidemics of 1970-1971. The introduction of a new R allele into the cultivar mixture provides a high degree of resistance in the beginning, but once the Avr phenotype emerges in the population the resistance breaks down. How can we break the boom-and-bust cycle and still use the R gene-mediated resistance? Well, the conclusion that a single R gene-mediated resistance is not durable shed light on the inherited resistance effects that has the properties of a quantitative trait. The advantage of the QR has shown to be more durable in an agricultural ecosystem that could be part of the long-

term solution. Burn et al. (2010) use the QR in a long-term experiment, which shows that the corresponding Avr phenotype emerged after three years in the population lacking the QR, meanwhile in the population carrying the QR shows no symptoms of the fungus infection. In contrast, the QR is hard to work with and has one disadvantage. The method is dependent on a normal distribution of multiple phenotypes. One interesting observation of the QR method is that it can be used to extend the life expectancy of these genes. Combining the QR method with other development strategies in a customized crop-breeding program may provide an environmentally friendly long-term solution to control plant diseases. It’s however easy to customize new development strategies on paper but difficult to implicate them in practice. Hypothetically speaking, if we design a gene development strategy using a combination of the methods discussed in this literature study starting with the gene rotation. The advantages of the gene rotation method are that R genes can be deployed over a larger agricultural landscape with a spatiotemporal rotation. This would avoid the typical bust that would normally occur of a single R gene-mediated resistance. This would still result in a short- term solution but combining several quantitative traits of these genes deployed in gene pyramids would extend the life expectancy that could be a part of a long-term solution. The degree of resistance in these gene pyramids varies depending on the combination of the R genes involved. A good example mentioned in this study presented by McCallum et al. (2016) shows how choosing a specific combination of R genes deployed in gene pyramid could maintain durable resistance from the year of 1991-2014. The use of multiple cultivar mixtures is also an interesting approach to achieve more sustainable and durable resistant plants due to the several degrees of resistance. Sapoukhina et al. (2013) show how wheat yellow rust decrease through the use of multiple cultivar mixtures containing R genes. The resistance efficiency within the wheat lines is enhanced by the addition of the R-mixture that provides a higher degree of resistance against wheat yellow rust. What we can learn today is that it’s time to use the knowledge of the past, use the present and breed for a more sustainable future. A single R gene-mediated resistance has its limitations and is therefore not durable in a long-term solution, in other hands the quantitative traits of these genes can be used in a sustainable way to be a part of the long-term solution in combination with other plant breeding programs. The involvement of multiple R gene-mediated resistance has shown the potential to be of great use as an environmentally friendly way to improve and control plant disease. It would be interesting to look in the near future at studies that implicate the knowledge of these genes in field studies with the aim to achieve durable and sustainable resistance plants in parts of the world where the local food production is at risk. More transgenic field studies, especially on staple foods that are widely consumed and provides great nutrition such as maize and rice. It would also be interesting to see more studies that have a molecular approach such as creating synthetic R genes.

ACKNOWLEDGMENTS I want to thank my supervisor, Mats Andersson who took time out to give me valuable comments and assisted me to carry out my degree thesis. Other special thanks to Daniel Carlberg, MoLab Kristianstad, for his previous comments and discussions that contributed to the improvement of the final version of this degree thesis. I want to thank Cornelia Spetea-Wiklund, for being the examiner of this degree thesis. I also want to take this opportunity to express my deepest gratitude and thanks to my family and friends for being a great support system during the writing weeks of this degree thesis as well as the Department of Biological and Environmental Science at the University of Gothenburg that made this thesis possible.

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APPENDIX 1, SEARCH SCHEDULE

Date Database Search nr. Keywords and Type of Number of Read Relevant Boolean operator search (e.g hits abstract articles (AND) abstract, free text) 2018 PubMed 1 R-genes AND NB- Free text 619 48 10 11 12 Central LRR 2018 PubMed 2 R-genes AND plant- Free text 1,080 37 5 11 14 Central pathogen interaction 2018 PubMed 3 Plant-breeding Free text 607 31 5 11 14 Central NB-LRR 2018 PubMed 4 Plant resistance R- Free text 2733 54 6 11 14 Central genes 2018 PubMed 5 Plant-pathogen Free text 67 12 2 11 17 Central interaction* 2018 PubMed 6 R genes AND plant Free text 5 3 1 11 17 Central resistance AND NB- LRR AND plant- breeding*