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Resistance screening and in-silico characterization of cloned novel RGA from multi race resistant Lentil germplasm against Fusarium wilt (Fusarium oxysporum f. sp. lentis)

Fusarium wilt caused by Fusarium oxysporum f. sp. lentis (Fol) is the most devastating disease of lentil present worldwide and in India. Identification of multi-race fusarium wilt resistance genes and incorporation into existing cultivar will help to reduce yield loss. In the present study, a hundred lentil germplasm were screened against seven prevalent races of Fol and accession IC201561, EC714243 and EC718238 were identified resistant. The typical R gene codes for the nucleotide-binding site and leucine-rich repeats (NBS-LRR) at the C terminal linked to either Toll/interleukin 1-like receptor (TIR) or coiled-coil (CC) at the N terminal. In the present study degenerate primers designed from the NBS region amplifying P-loop to GLPLA motif isolated forty-five resistance gene analogues (RGA) from identified resistant accessions. The sequence alignment identified both classes of RGA, TIR and non-TIR based on the presence of Aspartate (D) and Tryptophan (W) at the end of kinase motif respectively. The phylogenetic analysis grouped RGA into six classes, LRGA1 to LRGA6 determining the diversity of RGA present in the host. Grouping of RGA identified from Lens nigricans, LnRGA 2, 9, 13 with I2 reveals a probable role in Fusarium resistance. The similarity index of 27.85% to 86.98% was found among RGA and 26.83% to 49.41% between known R genes, I2, Gpa2, M and L6. Active binding sites present along the conserved motifs have grouped the RGA into 13 groups. ADP/ATP being the potential ligand determines ATP binding and ATP hydrolysis activity of RGA. The isolated RGA can be used in developing marker linked to the functional R gene. Further, expression analysis and full-length gene isolation further pave path to identifying the molecular mechanism involved in resistance.


Introduction 54
Lentil (Lens culinaris Medikus subsp. culinaris) is one of the most important cool-season legume food 55 crop grown after chickpea. It is an annual, self-pollinating diploid (2n= 14) crop having a genome size 56 of approximately 4Gb [1]. It is the oldest crop that originated in Turkey and is now cultivated in almost 57 all parts of the world. It is majorly grown in North America, Africa, Middle East and Asia. Globally 58 lentil is grown in an area of 6.1million ha and produce about 6.33mtons of yield. India has the largest 59 area under cultivation of approximately 1.51 mha and placed second in production after Canada 60 producing 1.096 million metric tons [2]. Lentil is a rich source of protein (24-26%) and fiber. It is also 61 a source of micronutrients such as calcium, phosphorus and iron and essential amino acids such as 62 lysine [3]. It is majorly grown as a rabi season legume crop in northern states of India such as Delhi, germplasm has been less explored. Screening for resistance during different plant growth stages helps 76 to identify late wilters during the reproductive stage and temporal variation of resistance [9]. Wild lentil 77 species have been identified as a potential source of resistance that can be tailored for resistance 78 breeding [10]. Multilocation screening of germplasm identifies germplasm resistant to multiple races 79 present in a geographic region [11]. Conventionally, the Resistance (R) gene is isolated by transposon Purified PCR product was ligated to pGEMT easy vector (Promega, Madison, Wis.) and cloned to E.

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coli JM109 according to manufactures protocol. About fifty positive colonies from each transformation 136 were screened using colony PCR. Clones producing a band of ͌ 510bp were further proceeded for plasmid 137 isolation and EcoR1 (Thermo Fisher) restriction digestion. Plasmids were isolated using Wizard Plus

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In-sillico characterization 141 Obtained sequences were trimmed for vector contamination and a similarity search was performed 142 using BLAST algorithm in GenBank database. The amino acid sequences were deduced using Expasy.

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Multiple alignment of obtained amino acids was carried out using CLUSTALX in BioEdit software.

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The tertiary structure of RGA was determined using I-TASSER software based on C-score, TM score 159 and RMSD (root mean square deviation) best-predicted tertiary structure was selected.

Result
Screening of germplasm against Fol races 162  One hundred accessions of lentil were screened against seven races of Fusarium wilt from seedling to   163  pod filling stage at 7 days interval for two cropping seasons, 2020-2021 and 2021-2022 and were graded   164 into 5 classes with scale of 1-9 based on disease incidence (DI). Accessions showed varying degrees of  (Tables 1and 2). Variation in the mean DI between races might be associated with the virulence of race 181 and differential interplay between host and race. The CV value of wild species showed large variation 182 due to less sample size and varied disease reactions within species (Figs 1 and 2). Few accessions 183 showed variation in disease incidence in two seasons might be due to varied environmental conditions 184 such as temperature.  response to Fol and were used for RGA isolation and characterization (S1 Figure). The degenerate 199 primers were designed based on conserved region of NBS amplifying P-loop to GLPLA region (S1 200  Table) and used for PCR amplification of genomic DNA of selected resistant accession, IC201561, 201 EC714243 and EC718238. Amplicon size of 510 bp was eluted and cloned to pGEMT vector and E.

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The phylogenetic tree was constructed using the Neighbour-joining method to determine the 225 relationship between obtained RGA and the known R gene. The resulting tree gave rise to two branches, with Fusarium R gene, I2 reveals its sequence homology and potential role in fusarium wilt resistance.

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Grouping of isolated RGAs to TIR and non-TIR classes reflects the diversity of RGA present in the 235 genus (Fig 5).   (Table 4).     Table). I-TASSER software was used to predict the active binding site of 277 RGA and its respective ligand. The identified active binding sites (ABS) were found to be present along LnRGA13 as active binding sites. ADP/ATP was found to be a potential ligand of RGAs with the 290 molecular function of ATP binding and ATPase activity (S5 Table). Previous reports of RNBS-A,

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RNBSC and GLPLA motif involved in ATP binding and hydrolysis has not been reported. Based on 292 amino acid corresponding to ABS RGAs were classified into thirteen groups (Table 5) probably due to change in single amino acid (Fig 8).

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LcRGA11 and LnRGA1 had the transmembrane region in the helix depicts probable interaction with 325 the lipid bilayer (Table 6). Best tertiary structure lentil RGA predicted based on C-score, TM-score and 326 RMSD using I-TASSER. The C-score of 45 lentil RGA ranged from 0.31 (LcRGA14 and LoRGA2) to  Table). Solubility of active binding site amino acid ranged from 1-4 revealing the buried 329 nature of ABS. It was predicted that RGA were isolated from genomic DNA represent constitutively 330 inactive/closed conformation in nature until pathogen attack (S5 Figure).

357
Higher CV has been observed in wild species due to diverse disease reaction and small sample size.

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Extensive screening has explored the potentiality of all species and subspecies of lentil against existing 359 races of Fol providing an excellent source for R gene isolation. To our best knowledge this is the first 360 report of multi-race resistance in lentil against Fusarium wilt.

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In last the decade PCR based approach for isolation of gene using degenerate primer has been identified