Cloning and Characterization of Osmotin (Osmst) Alleles from Potato Cultivar ‘Kufri Chipsona 1’

bioRxiv preprint doi: https://doi.org/10.1101/2020.01.03.894030; this version posted January 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Cloning and characterization of Osmotin (OsmSt) alleles from potato cultivar ‘Kufri Chipsona 1’

Amanpreet Kaur and Anil Kumar*

TIFAC-Centre of Relevance and Excellence in Agro and Industrial Biotechnology (CORE), Department of Biotechnology, Thapar Institute of Engineering & Technology, Patiala-47001, India *Correspondence: [email protected]

Abstract

The present study was focussed to clone and sequence characterise alleles of osmotin from

cDNA of Solanum tuberosum L. cultivar ‘Kufri Chipsona 1’. The genes vary in sizes as well

as were found to have eleven point mutations throughout the coding sequence. One deletion of

7 bp was also found in smaller form of the gene having molecular weight of 19.91 KDa.

Osmotin gene is known to impart resistance/tolerance to various fungal diseases in addition to

its role as an osmoprotectant, thus, cloned osmotin alleles from important processing grade

potato cultivar could become potential candidates for molecular breeding of potato.

Keywords: Potato, Pathogenesis related proteins, disease resistance

Introduction

Under the natural conditions, plants are continuously subjected to various biotic and abiotic

stresses causing impaired cellular growth (Kumar et al. 2015). To sustain these stresses, plants

have evolved a cascade of defense strategies involving release of various proteins such as

pathogenesis related (PR) proteins (Li et al. 2015). On the basis of their biochemical properties

and mechanism of action, PR proteins are divided into 17 families including β-1,3 glucanases

(PR-2), chitinases (PR-3, 4, 8 and 11), thaumatin-like proteins (PR-5), ribosome inactivating

proteins (PR-10), defensins (PR-12), thionins (PR-13) and lipid transfer proteins (PR-14) (Kaur

et al. 2017a). Among these, PR-5 protein family is known for its antimicrobial activity,

osmoregulation and role in plant development (Mani et al. 2012). PR-5 family is named as

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thaumatin-like protein (TLPs) due to its close similarity with sweet tasting protein thaumatin

of plant Thaumatococcus danielli (Velazhahan et al. 1999). PR-5 proteins are classified into

three types (acidic, neutral and basic) based on their isoelectric points (Kumar et al. 2015). Out

of all the known PR-5 proteins, osmotin or osmotin-like proteins (OLPs) are well known for

their role as osmoprotectants and anti-fungal agents (Chowdhury et al. 2015, 2017). They are

reported to get accumulated under stress conditions leading to ion/solute compartmentalization

and anti-phytopathogen activities (Kumar et al. 2015).

Osmotin, a multifacet stress inducible protein was firstly isolated from tobacco cells exposed

to saline conditions (Choi et al. 2013). The protein have been cloned from various plants such

as Nicotiana tabaccum (Lui et al. 1994, Evers et al. 1999, Zuker et al. 2001, Ouyang et al.

2005, Qin et al. 2006, Noori and Sokhansanj 2008, Parkhi et al. 2009, Goel et al. 2010, Rao et

al. 2011, Husaini et al. 2012, Subramanyam et al. 2011), Solanum nigrum (Vasavirama and

Kirti 2012), Capsicum annuum (Choi et al. 2013), Solanum tuberosum (Rivero et al. 2012),

etc. The over-expression of osmotin have been reported to exhibit anti-fungal activity against

diverse range of pathogens such as Puccinia triticina (Li et al. 2015), Fusarium graminearum

(Datta et al. 1999, Mackintosh et al. 2007), Phytophthora capsici, Fusarium oxysporum (Zuker

et al. 2001, Mani et al. 2012), Phytophthora infestans (Liu et al. 1994, 1996, Li et al. 1999),

Rhizoctonia solani (Rivero et al. 2012), etc. The anti-fungal property of the protein attributes

to its capability to bind to domains present on the fungal plasma membrane (Thevissen et al.

2005) which leads to dissipation of membrane integrity and can also inhibit spore germination

through spore lysis (Koiwa et al. 1997). Osmotin has been reported to be induced in plants

exposed to salt stress (Qureshi et al. 2007), cold stress (Patade et al. 2012), ethylene, wounding

(Zhu et al. 1995) and biotic stresses (Elvira et al. 2008, Mukherjee et al. 2010, Choi et al. 2013).

Due to its anti-fungal properties and highly stable nature, osmotin is now considered as a

natural food preservative (Thery et al. 2019). Osmotin also shows structural and functional

2 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.03.894030; this version posted January 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

similarity to insulin-sensitizing hormone, adinopectin, which further highlights the importance

of protein in therapeutics (Kumar et al. 2015).

In the present study, we report cloning and characterization of two osmotin alleles from an

important processing grade Indian potato cultivar ‘Kufri Chipsona 1’. The attempts were also

made to highlight characteristic features and structure of osmotin proteins encoded by the

cloned alleles using bioinformatics tools. The cloned gene can be used in molecular breeding

of potato and other plants to improve resistance/ tolerance against various abiotic and biotic

stress conditions.

Material and Methods

Plant material and chemicals

Microshoot cultures of Solanum tuberosum L. cultivar ‘Kufri Chipsona 1’ (CS-1) maintained

at plant tissue culture laboratory, Thapar Institute of Engineering & Technology, Patiala

through regular subculture on MS1 [basal Murashige and Skoog (1962) medium containing

10µM silver nitrate] medium (Kaur et al. 2017b) were used for cloning of osmotin (OsmSt)

gene. The cultures were incubated at 25±2 °C, under photoperiod duration of 16/8 h day/night

and photo intensity of 50 µmol.m2/s provided by cool fluorescent lamps (Philips Ltd., India).

All tissue culture grade chemicals and enzymes were purchased from HiMedia Laboratories

Ltd. (Mumbai, India) and ThermoScientific Laboratories Ltd. (Mumbai, India) respectively.

Cloning of OsmSt gene

Total RNA was isolated from the microshoots of potato cultivar CS-1 following CTAB method

(Chang et al. 1993) and cDNA was synthesized using RevertAid First Strand cDNA synthesis

kit (Fermentas Life Sciences, U.S.A) according to manufacturer’s guidelines. cDNA diluted

by 100 times was used as template for gene amplification using primers [Pair 1: Forward primer

5’-CGGGATCCCGCCACAAACATGGCCTACTT-3’ , Reverse Primer 5’-

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CGAGCTCGTTACTTAGCCTCTTCATCACTTGAAG-3’, Pair 2: Forward Primer 5’-

CGGGATCCGCCAGACGTGGGTCATAAAT-3’, RP 5’-

CGAGCTCGCGGGCCAGTAACACCATTAG-3’] having overhangs with restriction site for

BamHI and SacI. The PCR amplification was carried out in GenAmp 2700 thermocycler

(Applied Biosystem, USA) using reaction mixture consisting of template (cDNA), Taq

Polymerase (1 U), dNTPs mixture (100 µmol), 2 µl reaction buffer (10X), primers (10 nmol

each) and sterile Milli-Q water (Millipore India, Bangalore) to make up the volume upto 20 µl.

The amplification conditions includes initial denaturation at 94 ºC for 5 min followed by 31

cycles of 94 ºC (1 min), 60 ºC (45 sec), 72 ºC (1.5 min) and final extension at 72 ºC for 5 min.

The amplified DNA fragments separated on ethidium bromide stained agarose gel (1% w/v)

were visualised on U.V. transilluminator and documented using GelDoc system

(Biosystematica, U.S.A).

PCR products was gel purified using the PCR purification kit (Qiagen, USA) and restricted

using BamHI and Sac1 for directional cloning into pBI121 binary vector (Chen et al. 2003).

The ligated products were introduced into E. coli strain DH5α and propagated on Luria Agar

plates containing kanamycin (50 mg/l) as selection antibiotic. The modified plasmids were

isolated from the selected clones and sequenced in Applied Biosystems automatic sequencer

(DNA Sequencing Facility, Department of Biochemistry, South Campus, Delhi University,

New Delhi, India) using gene specific primers.

Analysis of sequence data

The sequences of osmotin alleles (OsmSt1 and OsmSt2) thus obtained were compared with

reported nucleotide and protein sequences those available in GenBank/ EMBL databases using

BLAST program (Altschul et al. 1997) accessible through NCBI (National Centre for

Biotechnology Information – http://www.ncbi.nlm.nih.gov). Multiple sequence alignments

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were performed using MULTALIN program (http://multalin.toulouse.inra.fr/multalin/). The

identification of the ORF and amino acid sequence coded by the cloned genes were deduced

using the ORF finder program provided by the NCBI

(http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Homology searches for deduced amino acid

sequence were performed with BLAST and multiple sequence alignments were carried out with

OsmSt1 and OsmSt2 protein sequences using CLUSTALW programme available online at

www.ebi.ac.uk. The alignment was modified to highlight conserved regions using GeneDoc

programme (www.nrbsc.org/gfx/genedoc). Potential transmembrane segments were identified

using TMHMM-2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0) programme. The

glycosylation and phophorylation sites were identified using NetNGlyc 1.0 and NetPhos 2.0

Server from technical University of Denmark (http://genome.cbs.dtu.dk//cgi-

bin/webface?jobid=netNglyc and http://genome.cbs.dtu.dk//cgi-bin/webface?jobid=netphos).

The molecular mass and isoeletric point of both proteins were computed using Compute pI/Mw

tool from ExPASy (Expert ProteinAnalysis System) bioinformatics resource portal

(http://web.expasy.org/compute_pi/) (Bjellqvist et al. 1993). Similarly for calculating the

amino acid composition of the predicted amino acid sequences, the ProtParam tool of ExPASy

proteomics server of the Swiss Institute of Bioinformatics (SIB; http://expasy.org/tools/) was

used. Domains and family of both proteins were identified using interproscan tool of European

Bioinformatics Institute (http://www.ebi.ac.uk/Tools/pfa/iprscan/) (Quevillon et al. 2005).

Molecular modelling and prediction of disulphide bridges

The amino acid sequences of OsmSt1 and OsmSt2 were first subjected to PSI-BLAST searches

(Altschul et al. 1997) to find homologous template. The single highest scoring template

providing maximum coverage with 100% confidence was used for modelling. Consequently,

the structure of PR-5d of N. tabaccum was retrieved and used to develop model of OsmSt1 and

OsmSt2 using Phre2 protein fold recognition server (www.sbg.bio.ic.ac.uk). The three

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dimensional structure was predicted through alignment of target sequence with template

sequence.

The presence of disulphide bonds and state of cysteine residues in OsmSt1 and OsmSt2 were

predicted using DiANNA 1.1 web server (clavius.bc.edu). The scores were produced for each

pair of cysteine in the amino acid sequences and Edmonds-Gabov maximum weight matching

algorithm was used to pair cysteine residues for formation of disulphide bonds (Ferre and Clote

2005).

Result and Discussion

Osmotin and its various isoforms have been cloned from many plants (Capelli et al. 1997, Pla

et al. 1998, Jami et al. 2007, Singh et al. 2017, Mani et al. 2012, Li et al. 2015). The presence

of osmotin isoforms in same plant have been previously reported in Piper colubrinum (Mani

et al. 2012). The two isoforms were reportedly distinct from each other in terms of size, pI

value and molecular structure. In the present study, we report cloning and sequence

characterization of osmotin alleles from an important Indian potato cultivar ‘Kufri Chipsona 1’

(CS-1).

cDNA synthesised using high quality, intact RNA (416.7 ng/μl; A260/280 2.09) isolated from

actively growing microshoots of CS-1 yielded amplification of 733bp for both OsmSt1 and

OsmSt2 alleles. BLAST analysis of the sequences revealed high similarity of PCR products

with the osmotin. More than 99% nucleotide sequence similarity was observed between

osmotin alleles from CS-1 and other Solanum species [S. tubersosum; XP_006358887.1

(99.45%), S. lycopersicum; NP_001296216.1 (99.45%), S. pennellii; XP_015083272.1

(99.45%)]. It have been previously reported that osmotin gene is highly conserved among the

Solanaceae family (Kumar et al. 2015) and slight variation in nucleotide level may be due to

presence of single nucleotide polymorphism (SNPs) in the sequence (Jami et al. 2007). It has

6 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.03.894030; this version posted January 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

also been reported that GC content is used for hierarchical classification of a species (Wayne

et al. 1987). The reported GC content for land plants ranges between 30–50% (Lynch 2007).

In case of cloned OsmSt1 and OsmSt2 genes, GC content was found as 46.52% which fall in

the reported range.

Figure 1: Sequence alignment of osmotin alleles cloned from potato cultivar ‘Kufri Chipsona 1’ showing point mutations (at 418th, 555th, 651st, 664th, 683rd, 685th, 688-692nd positions) and deletion between 674th and 681st position in coding sequence of Osmotin allele 1 (OsmSt1)

Alignment of OsmSt1 with OsmSt2 showed presence of single nucleotide polymorphism (at

418, 555, 651, 664, 683, 685, 688-692 positions of nucleotides) and a deletion between 674 to

681 bp (Figure 1). The open reading frame (ORF) of OsmSt1 was found to have 561 nucleotides

encoding 186 amino acids whereas OsmSt2 was slightly larger having 576 nucleotides

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encoding 192 amino acids. Although showing high amino acid sequence similarity, the

functional properties were found to vary between the OsmSt1 and OsmSt2 proteins due to

presence of additional six amino acids in OsmSt2. The calculated molecular mass of OsmSt1

was found to be 19.91 kDa with an isoelectric point (pI) of 4.81, whereas OsmSt2 was having

molecular mass of 20.55 KDa and was slightly acidic (pI 6.06).

Figure 2: Multiple sequence alignment of amino acid sequences of ‘Kufri Chipsona 1’ osmotin (OsmSt1 and OsmSt2) with osmotin (OSM) and osmotin-Like-proteins (OLP) from different taxas viz. Nicotiana tabaccum (OSM-Nt, CAA43854.1), Brassica juncea (OLP-Bj, AAS48588.1), Solanum nigrum (OLP-Sn, AAL87640.1), Capsicum annuum (OLP-Ca, NP_001311827.1), Oryza sativa (OSM-Os, AAB67852.1), Arabidopsis thaliana (OSM-At, CAA61411.1), Rosa roxburghii (OSM-Rr, ABC39615.1), Piper colubrinum (OSM-Pc, ABX71220.1), Solanum tuberosum (OSM-Solanu, QC092097.1). Black coloured highlighted sequences indicates the conserved regions of the protein.

PSI-BLAST with 1 iteration revealed high similarity of CS-1 osmotin with other species (Table

1, Figure 2). The Osmotin protein from these species is reported to play a role in plant defense

system against abiotic stress and fungal invasions (Castillo et al. 2005, Kumar et al. 2015).

Therefore, there is a strong possibility that OsmSt1 and OsmSt2 from CS-1 is likely to show

similar antifungal activities.

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Table 1: BLAST analysis of amino acid sequences Accession Description Amino acid E-Value number similarity (%) XP_006358887.1 Osmotin-like protein from Solanum 99.45 5e-131 tuberosum NP_001296216.1 Osmotin-like protein (TPM-1) from 99.45 7e-131 Solanum lycopersicum XP_015083272.1 Osmotin-like protein (OSML13) from 99.45 1e-130 Solanum pennellii AAU93853.1 Osmotin-like protein (A13) from Solanum 98.90 1e-129 phureja P50701.1 Osmotin-like protein (OSML13) from 98.90 1e-130 Solanum commersonii QC092097 Osmotin from Solanum tuberosum 97.84 3e-131 AAS48588.1 Putative Osmotin-like protein from 93.44 2e-124 Brassica juncea AAL87640.1 Osmotin-like protein from Solanum nigrum 93.96 2e-124 NP_001311827.1 Osmotin-like protein from Capsicum 93.89 2e-122 annuum CAA43854.1 Osmotin from Nicotiana tabacum 87.85 2e-113 CAA61411.1 Osmotin from Arabidopsis thaliana 70.43 2e-84 ABX71220.1 Osmotin from Piper colubrinum 75.46 3e-80

Both proteins were found to be acidic in nature with presence of more acidic amino acids in

comparison with basic amino acids (https://web.expasy.org/protparam/). It was also found that

some amino acids such as Gly (11.9-12.0%), Pro (9.7-9.9%), Ser (7.0-7.3%), Thr (8.6-8.9%),

Cys (7.8-8.1%) and Asn (6.8-7.0 %) occurred more frequently whereas other amino acids such

as Ala (4.3-4.7%), Arg (3.6-3.8%), Asp (5.2-5.9%), Gln (4.2-4.3%), Glu (2.6-2.7%), His (1.0-

1.6%), Leu (4.2-4.3%), Met (1.6%), Lys (1.6-3.6%) and Trp (1.6%) occurs less frequently. It

has been previously reported that overall amino acid composition of the protein have an

important role in evolution (King and Jukes 1969). Further, the quality and structure of protein

is also found to be dependent upon amino acid composition (Dyer 1971). Osmotin protein is

reported to be rich in cysteine residues scattered all over the sequence. These residues form

disulphide bridges which is a signature of PR-5 family proteins (Min et al. 2004, Jami et al.

2007). Generally, the presence of 16 cysteine residues have been reported in osmotin (Kumar

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et al. 2015), but in the present study, only 15 cysteine residues were present which lead to

formation of 7 disulphide bridge only (Figure 3). The presence of disulphide bridges is known

to provide high stability to the protein (Mani et al. 2012). The instability index (II) calculated

for OsmSt1 and OsmSt2 using ProtPram tool of Expasy server were found to be about 38.90

which indicates that the proteins are highly stable (https://web.expasy.org/protparam/).

Figure 3: Prediction of disulphide bridges between cysteine residues of proteins (OsmSt1 and OsmSt2) encoded by osmotin alleles cloned from potato cultivar ‘Kufri Chipsona 1’

Figure 4: Molecular modelling of Osmotin using PR-5 protein from N. tabaccum (TaxID:4097) as template. a- b) homology models of OsmSt1 and OsmSt2 respectively showing three domains of protein consisting of nine β-sheets and four α-helices connected by various loops c) homology model of template showing typical structure of thaumatin-like-protein consisting of twelve β-sheets and four α-helices. Homology models of OsmSt1 and OsmSt2 are shown in Figure 4. The structures are typical to

PR-5 family proteins with three distinct domains. OsmSt1 and OsmSt2 were found to be similar

to each other having nine β-sheets and four α-helices (Figure 4a-b). The models were produced

using PR-5d protein from N. tabacum (d1auna_b.25.1.1; Tax ID 4097) as template which

provide 89% and 86% coverage (166 amino acids) to OsmSt1 and OsmSt2 repectively with

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100% confidence. The model of template was also produced (Figure 4c) which was also found

to have three distinct domains with 12 β-sheets and four α-helices. Our predicted models are in

line with the structural information of PR-5 family proteins (zaematin, thaumatin-like protein,

Osmotin) previously reported (Mani et al. 2012).

Figure 5: Prediction of phosphorylation sites in osmotin proteins (OsmSt1 and OsmSt2) using NetPhos 2.0 server (http://genome.cbs.dtu.dk//cgi-bin/webface?jobid=netphos) Glycosylation and phosphorylation are two important post-translational modification events

which modify protein properties by altering its distribution, folding and stability (Ciesla et al.

2011, Haltiwanger and Lowe 2004). Thus, in the present study, presence of glycosylation and

phosphorylation sites were analysed using NetPhos 2.0 and NetGlyc 1.0 servers. OsmSt1 and

OsmSt2 from CS-1 do not show the presence of glycosylation sites whereas the presence of 16

phosphorylation sites were observed (Figure 5). Similar findings were observed from reported

Osmotin-like protein of Solanum tuberosum accession XM_006358825.2. Out of 16 detected

phosphorylation sites, 11 were at serine residue, 4 at threonine residue and 1 at tyrosine residue

(Figure 5). These results were in line with previous findings which suggested the occurance of

phosphorylation on serine residues as most common phenomena (Thomason and Kay 2000). It

has also been reported that during interaction with fungal pathogen, osmotin utilizes signal

transduction pathway which is dependent on phosphorylation and dephosphorylation events

(Kim et al. 2002).

In conclusion, alleles of osmotin were cloned and characterized from Solanum tuberosum

cultivar ‘Kufri Chipsona 1’. The proteins encoded (OsmSt1 and OsmSt2) by these alleles were

found to be highly similar but posses different physio-chemical properties (pI and molecular

weight) which could determine their mode of action. The structural analysis of proteins

revealed presence of three distinct domains and various disulphide bridges which is a

characteristic feature of osmotin. The cloned alleles from CS-1 could be a candidate for in-

depth functional and evolutionary studies and could lead to potential molecular breeding

programmes for generation of disease resistant/ salt tolerant potato varieties.

Acknowledgement Amanpreet Kaur is thankful to University Grants Commission (UGC),

New Delhi for the award of Maulana Azad National Fellowship for minority students. TIFAC-

CORE is thanked for the facilities to carry out research work.

Author contribution Amanpreet Kaur conducted the experiments, compiled data, analyzed

the results and wrote the initial draft of the manuscript, Amanpreet Kaur and Anil Kumar

conceived and designed the experiments, Anil Kumar finalized the manuscript.

References Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389-3402. http://doi.org/ 10.1093/nar/25.17.3389 Bjellqvist B, Hughes GJ, Pasquali CH, Paquet N, Ravier F, Sanchez JC, Frutiger S, Hochstrasser D (1993) The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis 14:1023-1031. http://doi.org/ 10.1002/elps.11501401163 Capelli N, Diogon T, Greppin H, Simon P (1997) Isolation and characterization of cDNA clone encoding an osmotin-like protein from Arabidopsis thaliana. Gene 191:51–56. http://doi.org/10.1016/S0378-1119(97)000292 Castillo Ruiz RA, Ghislain CHM, Gebhardt C (2005) Organization of phenylalanine ammonia lyase (PAL), acidic PR-5 and Osmotin-like (OSM ) defence-response gene families in the potato genome. Mol Gen Genomics 274:168-179. http://doi.org/ 10.1007/s00438-005-0006-7

Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11:113-116. https://doi.org/10.1007/BF02670468 Chen P-Y, Wang C-K, Soong S-C, To K-Y (2003) Complete sequence of the binary vector pBI121 and its applications in cloning T-DNA insertion from transgenic plants. Mol Breed 11:287- 293. https://doi.org/10.1023/A:1023475710642. Choi DS, Hong JK, Hwang BK (2013) Pepper Osmotin-like protein 1 (CaOSM1) is an essential component for defense response, cell death, and oxidative burst in plants. Planta 238:1113– 1124. https://doi.org/10.1007/s00425-013-1956-3 Chowdhury S, Basu A, Kundu S (2015) Cloning, characterization and bacterial over-expression of an osmotin-Like protein gene from Solanum nigrum L. with antifungal activity against three necrotrophic fungi. Mol Biotechnol 57:371–381. https://doi.org/10.1007/s12033-014- 9831-4 Chowdhury S, Basu A, Kundu S (2017) Overexpression of a New Osmotin-Like Protein Gene (SindOLP) Confers tolerance against biotic and abiotic stresses in Sesame. Front Plant Sci 8:410. https://doi.org/10.3389/fpls.2017.00410 Ciesla J, Fraczyk T, Rode W (2011) Phosphorylation of basic amino acid residues in proteins: important but easily missed. Acta Biochimica Polonica 58:137-147 Datta K, Velazhahan R, Oliva N, Ona I, Mew T, Khush GS, Mew T, Khush GS, Muthukrishnan S, D atta SK. (1999) Over-expression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease. Theor Appl Genet. 98: 1138–1145. https://doi.org/10.1007/s001220051178 Dyer KF (1971) The quiet revolution: A new synthesis of biological knowledge. J Biol Educ 5:15-24. https://doi.org/10.1080/00219266.1971.9653663 Elvira MI, Galdeano MM, Gilardi P, García-Luque I, Serra MT (2008) Proteomic analysis of pathogenesis related pro-tein (PRs) induced by compatible and incompatible interactions of pepper mild mottle virus (PMMoV) in Capsicum chinense L3 plants. J Exp Bot 59:1253-1265. https://doi.org/10.1093/jxb/ern032 Evers D, Overney S, Simon P, Greppin H, Hausman JF (1999) Salt tolerance of Solanum tuberosum L. overexpressing an heterologous osmotin-like protein. Biol. Plant 42:105–112. http://doi.org/ 10.1023/A:1002131812340 Ferre F, Clote P (2005) DiANNA: a web server for disulphide connectivity Prediction. Nucleic Acids Res 1;33 (Web Server issue):W230-2. http://doi.org/ 10.1093/nar/gki412 Goel D, Singh AK, Yadav V, Babbar SB, Bansal KC (2010) Overexpression of osmotin gene confers tolerance to salt and drought stresses in transgenic tomato (Solanum lycopersicum L.). Protoplasma 245:133–141. http://doi.org/10.1007/s00709-010-0158-0 Haltiwanger RS, Lowe JB (2004) Role of Glycosylation in Development. Annu Rev Biochem 73:491- 537. http://doi.org/ 10.1146/annurev.biochem.73.011303.074043 Husaini AM, Abdin MZ, Khan S, Xu YW, Aquil S, Anis M (2012) Modifying strawberry for better adaptability to adverse impact of climate change. Curr Sci 102:1660–1673 Jami SK, Anuradha TS, Guruprasad L, Kirti PB (2007) Molecular, biochemical and structural characterization of osmotin-like protein from black nightshade (Solanum nigrum). J Plant Physiol 164:238–252. http://doi.org/ 10.1016/j.jplph.2006.01.006 Kaur A, Kumar A, Reddy MS (2017a) Plant-pathogen interactions- A proteomic approach. In Singh R, Kothari R, Koringa PG and Singh SP (eds) Understanding host-microbiome interactions- An omics Approach. Springer pp. 207-227. http://doi.org/10.1007/978-981-10-5050-3 Kaur A, Reddy MS, Kumar A (2017b) Efficient, one step and cultivar independent shoot organogenesis of potato. Physiology and Molecular Biology of Plants 23:461-469. https://doi.org/10.1007/s12298-017-0418-y

Kim H, Mun JH, Byun H, Hwang H-J, Kwon YM, Kim S-G (2002) Molecular cloning and characterization of the gene encoding Osmotin protein in Petunia hybrida. Plant Sci 162:745- 752. https://doi.org/10.1016/S0168-9452(02)00016-X King JL, Jukes TH (1969) Non- Darwinian evolution. Science 164:788-798 Koiwa H, Kato H, Nakatsu T, Oda J, Yamada Y, Sato F (1997) Purification and characterisation of tobacco pathogenesis-related protein PR-5d, an anti-fungal thaumatin-like protein. Plant Cell Physiol 38:783-791. https://doi.org/10.1093/oxfordjournals.pcp.a029236 Kumar SA, Kumari PH, Kumar GS, Mohanalatha C, Kishor PBK (2015) Osmotin: a plant sentinel and a possible agonist of mammalian adiponectin. Front Plant Sci 6:163. http://doi.org/10.3389/fpls.2015.00163 Li R, Wu N, Fan Y, Song B (1999) Transgenic potato plants expressing osmotin gene inhibits fungal development in inoculated leaves. Chin J Biotechnol 15:71–75 Li X-Y, Gao L, Zhang W-H, Liu J-K, Zhang Y-J, Wang H-Y, Liu D-Q (2015) Characteristic expression of wheat PR5 gene in response to infection by the leaf rust pathogen, Puccinia triticina. J Plant Interact 10:132-141. https://doi.org/10.1080/17429145.2015.1036140 Liu D, Raghothama KG, Hasegawa PM, Bressan RA (1994) Osmotin overexpression in potato delays development of disease symptoms. Proc. Natl. Acad. Sci. U.S.A.91:1888–1892. http://doi.org/ 10.1073/pnas.91.5.1888 Liu D, Rhodes D, D’Urzo MP, Xu Y, Narsimhan ML, Hasegawa PM, et al. (1996) In vivo and in vitro activity of truncated osmotin that is secreted into the extracellular matrix. Plant Sci 121:123– 131. http://doi.org/10.1016/S0168-9452(96)04514-1 Lynch M (2007) The Origins of Genome Architecture, Ed. 1. Sinauer Associates, Sunderland, MA Mackintosh CA, Lewis J, Radmer LE, Shin S, Heinen SJ, Smith LA, Wyckoff MN, Dill-Macky R, Evans CK, Kravchenko S, Baldridge GD, Zeyen RJ, Muehlbauer GJ (2007) Overexpression of defense response genes in transgenic wheat enhances resistance to Fusarium head blight. Plant Cell Rep. 26:479–488. https://doi.org/10.1007/s00299-006-0265-8 Mani T, Sivakumar KC, Manjula S (2012) Expression and Functional Analysis of Two Osmotin (PR5) Isoforms with Differential Antifungal Activity from Piper colubrinum: Prediction of Structure–Function Relationship by Bioinformatics Approach. Mol Biotechnol 52:251-261. https://doi.org/ 10.1007/s12033-011-9489-0 Min K, Ha SC, Hasegawa PM, Bressan RA, Yun DJ, Kim KK (2004) Crystal structure of Osmotin, a plant antifungal protein. Proteins 54:170-173. http://doi.org/ 10.1002/prot.10571 Mukherjee AK, Carp MJ, Zuchman R, Ziv T, Horwitz B, Gepstein S (2010) Proteomics of the response of Arabidopsis thaliana to infection with Alternaria brassicicola. J Proteom73:709–720. http://doi.org/ 10.1016/j.jprot.2009.10.005 Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x Noori SAS, Sokhansanj A (2008) Wheat plants containing an osmotin gene show enhanced ability to produce roots at high NaCl concentration. Russ J Plant Physiol 55:256–258 http://doi.org/ 10.1134/S1021443708020143 Ouyang B, Chen YH, Li HX, Qian CJ, Huang SL, Ye ZB (2005) Transformation of tomatoes with osmotin and chitinase genes and their resistance to Fusarium wilt. J Horticult Sci Biotechnol 80:517–522. Parkhi V, Kumar V, Sunilkumar G, Campbell LAM, Singh NK, Rathore KS (2009) Expression of apoplastically secreted tobacco osmotin in cotton confers drought tolerance. Mol Breed 23, 625–639. doi: 10.1007/s11032-009-9261-3 Patade VY, Khatri D, Manoj K, Kumari M, Ahmed Z (2012) Cold tolerance in thiourea primed Capsicum seedlings is associated with transcript regulation of stress responsive genes. Mol Biol Rep 39: 10603–10613. http://doi.org/10.1007/s11033-012-1948-6

Pla M, Huguet G, Verdaguer D, Puigderrajols P, Llompart B, Nadal A, et al. (1998) Stress proteins co-expressed in suberized and lignified cells and in apical meristems. Plant Sci 139:49–57. http://doi.org/10.1016/S0168-9452(98)00169-1 Qin J, Zuo K, Zhao J, Ling H, Cao Y, Qiu C, et al. (2006) Overexpression of GbERF confers alteration of ethylene-responsivegene expression and enhanced resistance to Pseudomonas syringae in transgenic tobacco. J BioSci 31: 255–263. http://doi.org/10.1007/BF02703918 Quevillon E, Silventoinen V, Pillai S, Harte N, Mulder N, Apweiler R, Lopez R (2005) InterProScan: protein domains identifier. Nucleic Acids Res 33:W116-W120. http://doi.org/ 10.1093/nar/gki442 Qureshi MI, Qadir S, Zoll L (2007) Proteomics based dissection of stress-responsive pathways in plants. Plant Physiol.164, 1239–1260. http://doi.org/10.1016/j.jplph.2007.01.013 Rao MVR, Parameswari C, Sripriya R, Veluthambi K (2011) Transgene stacking and marker elimination in transgenic rice by sequential Agrobacterium-mediated co-transformation with the same selectable marker gene. Plant Cell Rep 30, 1241–1252. http://doi.org/10.1007/s00299-011-1033-y Rivero M, Furman N, Mencaccia N, Picca P, Toum L, Lentz E ,et al. (2012) Stacking of antimicrobial genes in potato transgenic plants confers increased resistance to bacterial and fungal pathogens. J. Biotechnol 157:334–343. http://doi.org/10.1016/j.jbiotec.2011.11.005 Singh V (2017) Biotechnological interventions against leaf spot disease of Withania somnifera (L.) Dunal. Ph.D. Thesis, Guru Nanak Dev University, Amritsar Subramanyam K, Sailaja KV, Subramanyam K, Rao DM, Lakshmidevi K (2011) Ectopic expression of an osmotin gene leads to enhanced salt tolerance in transgenic chilli pepper (Capsicum annum L.). Plant Cell Tissue Organ. Cult. 105:181–192. http://doi.org/10.1007/s11240-010- 9850-1 Thery T, Lynch KM, Arendt EK (2019) Natural Antifungal Peptides/Proteins as Model for Novel Food Preservatives. Comprehensive Reviews in Food Science and Food Safety 18:1327-1360. http:// doi.org/10.1111/1541-4337.12480 Thevissen K, Idkowiak-Baldys J, Im YJ, Takemoto J, Francois IE, Ferket KK, Aerts AM, Meert EM, Winderickx J, Roosen J, Cammue BP (2005) SKN1, a novel plant defensin-sensitivity gene in Saccharomyces cerevisiae, is implicated in sphingolipid biosynthesis. FEBS Lett 579:1973– 1977 Thomason P and Kay R (2000) Eukaryotic signal transduction via histidine- aspartate phosphorelay. J Cell Sci 113:3141-3150 Vasavirama K, Kirti PB (2012) Increased resistance to late leaf spot disease in transgenic peanut using a combination of PR genes. Funct Integr Genomics 12:625–634. http://doi.org/10.1007/s10142-012-0298-8 Velazhahan R, Datta SK, Muthukrishnan S (1999) The PR-5 family: thaumatin-like proteins in plants. In: Datta SK, Muthukrishnan S, editors. Pathogenesis-related proteins in plants. Boca Raton, FL:CRCpress;p 107-29 Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O, Krichevsky MI, Moore LH, Moore WEC, Moore RGE, Stackebrandt E, Starr MP, Truper HG (1987) International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37:463-464. 0020-77 13/87/040463-02$02.00/0 Zhu B, Chen THH, Li PH (1995) Activation of two osmotin-like protein genes by abiotic stimuli and fungal pathogen in transgenic potato plants. Plant Physiol 108:929–937. http://doi.org/10.1104/pp.108.3.929 Zuker A, Shklarman E, Scovel G, Ben-Meir H, Ovadis M, Neta-Sharir I, et al. (2001) Genetic engineering of agronomic and ornamental traits in carnation. Acta Horticul 560:91–94

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