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

Abstract

Fusarium wilt caused by Fusarium oxysporum f. sp. lentis (Fol) is the most devastating disease of lentil present worldwide. Identification of multi-race fusarium wilt resistance genes and their incorporation into existing cultivars will help to reduce yield losses. In the present study, 100 lentil germplasms belonging to seven lentil species were screened against seven prevalent races of Fol, and accessions IC201561 (Lens culinaris subsp. culinaris), EC714243 (L. c. subsp. odemensis), and EC718238 (L. nigricans) were identified as resistant. The typical R gene codes for the nucleotide-binding site and leucine-rich repeats (NBS-LRR) at the C terminal are linked to either the 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 the P-loop to the GLPLA motif, isolated forty-five resistance gene analogues (RGAs) from identified resistant accessions. The sequence alignment identified both classes of RGAs, TIR and non-TIR, based on the presence of aspartate (D) and tryptophan (W) at the end of the kinase motif, respectively. The phylogenetic analysis grouped the RGAs into six classes, from LRGA1 to LRGA6, which determined the diversity of the RGAs present in the host. Grouping of the RGAs identified from Lens nigricans, LnRGA 2, 9, 13 with I2 revealed the structural similarity with the fusarium resistance gene. The similarity index ranged from 27.85% to 86.98% among the RGAs and from 26.83% to 49.41% among the known R genes, I2, Gpa2, M, and L6. The active binding sites present along the conserved motifs grouped the RGAs into 13 groups. ADP/ATP, being the potential ligand, determines the ATP binding and ATP hydrolysis activity of the RGAs. The isolated RGAs can be used to develop markers linked to the functional R gene. Furthermore, expression analysis and full-length gene isolation pave the path to identifying the molecular mechanism involved in resistance.

Keywords: cloning; fusarium wilt; in silico characterization; lentil; resistance gene analogue; screening.

Figure 1

Box plot depicting disease incidence of each species of lentil against each race of Fusarium oxysporum f. sp. lentis screened during 2020-2021. Sp1: Lens culinaris sub sp. culinaris; Sp2, L. c. subsp. tomentosus; Sp3, L. c sub sp. orientalis; Sp 4, L. c. sub sp. odemensis; Sp 5, L. lamottei; Sp 6, L. nigricans; Sp 7, L. ervoides. Race 1: MP-2; race 2, UP-9; race 3, RJ-8; race 4, DL-1; race 5, CG-5; race 6, UP-12; race 7, BR-27.

Figure 2

Box plot depicting disease incidence of each species of lentil against each race of Fusarium oxysporum f. sp. lentis screened during 2021-2022. Sp1, Lens culinaris sub sp. culinaris; Sp2, L. c. subsp. tomentosus; Sp 3, L. c sub sp. orientalis; Sp 4, L. c. sub sp. odemensis; Sp 5, L. lamottei; Sp 6, L. nigricans; Sp 7, L. ervoides. Race 1, MP-2; race 2, UP-9; race 3, RJ-8; race 4, DL-1; race 5, CG-5; race 6, UP-12; race 7, BR-27.

Figure 3

Pot evaluation of lentil accessions, (A) IC201561 (L65); (B) EC714243 (L83), (C) EC718238 (L90); and (D) L-9-12 (Susceptible check) against seven races of Fusarium oxysporum (f) sp. lentis. Pot in the left-hand corner is control followed by race 1 (MP-2), race 2 (UP-9), race 3 (RJ-8), race 4 (DL-1), race 5 (CG-5), race 6 (UP-12), and race 7 (BR-7).

Figure 4

PCR amplification product generated using LRGAF and LRGAR degenerate primer on genomic DNA of lentil resistant accession. Lane 1 IC201561 (L65), Lane 2 EC714243 (L83), and Lane 3 EC718238 (L90). M represents 100 bp ladder.

Figure 5

Multiple sequence alignment between P-loop and GLPLA of 45 lentil RGAs with NBS region of known R gene Gpa2, I2, N, L6, and M. The conserved motifs are indicated above the alignment. The number of amino acids is indicated above the alignment. The gaps to optimize the alignment are designated by dash (-). The alignment was constructed using CLUSTA W of Bioedit.

Figure 6

Neighbor-joining phylogenetic tree constructed based on isolated Lentil RGAs and known NBS region of R gene, N (U15605), L6 (U27081), M (U73916), RPP5 (AAFO8790.1), RPP4 (AAM18462.1), RPP1 (AT3444670), RPS4 (CAB50708.1), Mla (AAG37356), Pi-ta (ACY24970.1), Pi36 (ADF29629.1), Pib (BAA76282.2), I2 (AF004878), RPP13 (AAF42831), RPM1 (AQ39214), Prf (U65391), Gpa2 (AF195939), RPP8 (AAC78631.1.), FOM-2 (AY583855.1) at bootstrap values (10000 replicates). Numbers on the branches indicate the percentage of bootstrap replications. Green represents LRGA 1, blue represents LRGA 2, yellow represents LRGA 3, red represents LRGA 4, purple represents LRGA 5, and orange represents LRGA 6.

Figure 7

Neighbor-joining phylogenetic tree constructed based on isolated lentil RGAs, lentil R gene, and known NBS region of R genes N (U15605), L6 (U27081), M (U73916), RPP5 (AAFO8790.1), RPP4 (AAM18462.1), RPP1 (AT3444670), RPS4 (CAB50708.1), Mla (AAG37356), Pi-ta (ACY24970.1), Pi36 (ADF29629.1), Pib (BAA76282.2), I2 (AF004878), RPP13 (AAF42831), RPM1 (AQ39214), Prf (U65391), Gpa2 (AF195939), RPP8 (AAC78631.1.), and FOM-2 (AY583855.1) at bootstrap values (1000 replicates). Numbers on the branches indicate the percentage of bootstrap replications. Green represents LRGA 1, blue represents LRGA 2, yellow represents LRGA 3, red represents LRGA 4, purple represents LRGA 5, and orange represents LRGA 6.

Figure 8

Diagrammatic representation of conserved motif of TIR RGAs within NBS domain along with R genes N, L6, and M. The solid black line represents each RGA, and their lengths with motifs are represented in colored boxes. The sequence logo of six conserved motifs along with their E value is presented on the right-hand side.

Figure 9

Diagrammatic representation of conserved motif of non-TIR RGAs within NBS domain along with R genes Gpa2 and I2. The solid black line represents each RGA, and their lengths with motifs are represented in colored boxes. Sequence logo of six conserved motifs along with their E value is given below.

Figure 10

Phylogenetic tree constructed based on amino acid corresponding to active binding sites (ABS) using maximum likelihood in MEGA X with 1000 bootstrap replication. Lentil RGAs with the same color represent having a similar ABS. Blue represents GTDN, green represents GTD, black represents GTDG, red represents GTDT, yellow represents GTDL, orange represents GT, red represents L, pink represents GTS, light blue represents SN, purple represents G, light green represents GTNS, and gray represents S.