Ganesan Prakash

Cenchrus americanus (L.) Morrone (Poaceae), is an important millet crop cultivated mainly in arid and semiarid regions and is a staple food grain for millions of people. During 2021 July surveys in the pearl millet fields in Mysore (12°30'55" N; 76°56'54" E), Karnataka, India, plants showed spathe blight and leaf spot disease with an overall incidence ranging from 5 - 8% in the 15 hectares surveyed. Infected leaves appeared brown, and lesions extended to the sheath. Some spathes were also found infected with similar symptoms. Diseased leaves and spathes were collected (n = 5 each) for pathogen identification. Samples were cut into small pieces (0.5 cm2), sterilized with sodium hypochlorite (2%, v/v), and blotted dried. The associated fungal pathogen was isolated on potato dextrose agar (PDA) medium amended with Streptomycin (40 mg/L) and incubated at 28 ºC for 1 week. Colonies were grey, fluffy, cottony with an irregular margin, undulate and dark brown in the back of the plate. Conidiophores were pale brown, erect, slightly curved, septate, unbranched, verruculose and measured 27.1 - 94 µm in length × 2.3 - 4.5 µm in width (n = 20). Conidiogenous cells were brown, subcylindrical, irregularly shaped, and conidia were straight, mainly elliptical, dark brown smooth, with two to three septa, with measurements of 11.1 - 26.4 µm by 5.7 - 14.3 µm (n = 50). Based on morphological characters, the pathogen was identified as Curvularia sp. Two representative isolates (UOMPM1 & UOMPM2) were molecularly identified. The total genomic DNA was extracted with a CTAB method, and ITS, GAPDH and tef-1α loci were amplified using primers ITS1/ITS4 (White et al., 1990), GPD1/GPD2 (Berbee et al., 1999) and EF1983F/EF-2218R (Schoch et al., 2009) respectively. ITS sequence had 100% similarity (706/706bp) with reference sequence C. spicifera (MH863648; HF934915 & HF934916); tef-1α sequence had 100% (933/933bp) identity with C. spicifera (KM062878, KJ939505), and the GAPDH sequence was 99.8% identical to that of Curvularia sp. (MG979055), and C. spicifera (MH809681). Combined dataset of concatenated sequence (ITS-GAPDH-tef-1α) was used in a phylogenetic analysis and revealed that the isolates were in a common clade with the isolate of Curvularia spicifera (CBS 274.52) thus, confirming the identity of the isolated pathogen as C. spicifera. The sequences obtained in the present study were deposited in the GenBank (ITS: OQ253406, OQ253407; LSU: OQ253429, OQ253430; GAPDH: OQ263372, OQ263373 & TEF: OQ263374, OQ263375). Pathogenicity test was carried out by inoculating (foliar /whole plant spray) 60 healthy pearl millet plants (45-days old), grown in field plot with spore suspension (105 conidia/ml). Control plants (n=20) were treated with sterile water. The experiments were conducted in triplicates and repeated twice. Development of disease symptoms was recorded on 41 plants, and all control plants remained healthy. The identity was confirmed after re-isolation as C. spicifera based on cultural and molecular sequence analysis. To our knowledge, this is the first report of C. spicifera causing a leaf spot and spathe blight disease of pearl millet in India. This disease seriously affects grain production, and effective disease management strategies need to be investigated.

Main conclusion The investigation is the first report on genome-wide identification and characterization of NBLRR genes in pearl millet. We have shown the role of gene loss and purifying selection in the divergence of NBLRRs in Poaceae lineage and candidate CaNBLRR genes for resistance to Magnaporthe grisea infection. Abstract Plants have evolved multiple integral mechanisms to counteract the pathogens' infection, among which plant immunity through NBLRR (nucleotide-binding site, leucine-rich repeat) genes is at the forefront. The genome-wide mining in pearl millet (Cenchrus americanus (L.) Morrone) revealed 146 CaNBLRRs. The variation in the branch length of NBLRRs showed the dynamic nature of NBLRRs in response to evolving pathogen races. The orthology of NBLRRs showed a predominance of many-to-one orthologs, indicating the divergence of NBLRRs in the pearl millet lineage mainly through gene loss events followed by gene gain through single-copy duplications. Further, the purifying selection (Ka/Ks < 1) shaped the expansion of NBLRRs within the lineage of pear millet and other members of Poaceae. Presence of cis-acting elements, viz. TCA element, G-box, MYB, SARE, ABRE and conserved motifs annotated with P-loop, kinase 2, RNBS-A, RNBS-D, GLPL, MHD, Rx-CC and LRR suggests their putative role in disease resistance and stress regulation. The qRT-PCR analysis in pearl millet lines showing contrasting responses to Magnaporthe grisea infection identified CaNBLRR20, CaNBLRR33, CaNBLRR46 CaNBLRR51, CaNBLRR78 and CaNBLRR146 as putative candidates. Molecular docking showed the involvement of three and two amino acid residues of LRR domains forming hydrogen bonds (histidine, arginine and threonine) and salt bridges (arginine and lysine) with effectors. Whereas 14 and 20 amino acid residues of CaNBLRR78 and CaNBLRR20 showed hydrophobic interactions with 11 and 9 amino acid residues of effectors, Mg.00g064570.m01 and Mg.00g006570.m01, respectively. The present investigation gives a comprehensive overview of CaNBLRRs and paves the foundation for their utility in pearl millet resistance breeding through understanding of host–pathogen interactions.

Background: The nucleotide binding site leucine rich repeat (NBLRR) genes significantly regulate defences against phytopathogens in plants. The genome-wide identification and analysis of NBLRR genes have been performed in several species. However, the detailed evolution, structure, expression of NBLRRs and functional response to Magnaporthe grisea are unknown in finger millet (Eleusine coracana (L.) Gaertn.). Results: The genome-wide scanning of the finger millet genome resulted in 116 NBLRR (EcNBLRRs1-116) encompassing 64 CC-NB-LRR, 47 NB-LRR and 5 CC_R-NB-LRR types. The evolutionary studies among the NBLRRs of five Gramineae species, viz., purple false brome (Brachypodium distachyon (L.) P.Beauv.), finger millet (E. coracana), rice (Oryza sativa L.), sorghum (Sorghum bicolor L. (Moench)) and foxtail millet (Setaria italica (L.) P.Beauv.) showed the evolution of NBLRRs in the ancestral lineage of the target species and subsequent divergence through gene-loss events. The purifying selection (Ka/Ks < 1) shaped the expansions of NBLRRs paralogs in finger millet and orthologs among the target Gramineae species. The promoter sequence analysis showed various stress- and phytohormone-responsive cis-acting elements besides growth and development, indicating their potential role in disease defence and regulatory mechanisms. The expression analysis of 22 EcNBLRRs in the genotypes showing contrasting responses to Magnaporthe grisea infection revealed four and five EcNBLRRs in early and late infection stages, respectively. The six of these nine candidate EcNBLRRs proteins, viz., EcNBLRR21, EcNBLRR26, EcNBLRR30, EcNBLRR45, EcNBLRR55 and EcNBLRR76 showed CC, NB and LRR domains, whereas the EcNBLRR23, EcNBLRR32 and EcNBLRR83 showed NB and LRR somains. Conclusion: The identification and expression analysis of EcNBLRRs showed the role of EcNBLRR genes in assigning blast resistance in finger millet. These results pave the foundation for in-depth and targeted functional analysis of EcNBLRRs through genome editing and transgenic approaches.

Pusa Basmati 1509 (PB1509) is one of the major foreign-exchange-earning varieties of Basmati rice; it is semi-dwarf and early maturing with exceptional cooking quality and strong aroma. However, it is highly susceptible to various biotic stresses including bacterial blight and blast. Therefore, bacterial blight resistance genes, namely, xa13 + Xa21 and Xa38, and fungal blast resistance genes Pi9 + Pib and Pita were incorporated into the genetic background of recurrent parent (RP) PB1509 using donor parents, namely, Pusa Basmati 1718 (PB1718), Pusa 1927 (P1927), Pusa 1929 (P1929) and Tetep, respectively. Foreground selection was carried out with respective gene-linked markers, stringent phenotypic selection for recurrent parent phenotype, early generation background selection with Simple sequence repeat (SSR) markers, and background analysis at advanced generations with Rice Pan Genome Array comprising 80K SNPs. This has led to the development of Near isogenic lines (NILs), namely, Pusa 3037, Pusa 3054, Pusa 3060 and Pusa 3066 carrying genes xa13 + Xa21, Xa38, Pi9 + Pib and Pita with genomic similarity of 98.25%, 98.92%, 97.38% and 97.69%, respectively, as compared to the RP. Based on GGE-biplot analysis, Pusa 3037-1-44-3-164-20-249-2 carrying xa13 + Xa21, Pusa 3054-2-47-7-166-24-261-3 carrying Xa38, Pusa 3060-3-55-17-157-4-124-1 carrying Pi9 + Pib, and Pusa 3066-4-56-20-159-8-174-1 carrying Pita were identified to be relatively stable and better-performing individuals in the tested environments. Intercrossing between the best BC₃F₁s has led to the generation of Pusa 3122 (xa13 + Xa21 + Xa38), Pusa 3124 (Xa38 + Pi9 + Pib) and Pusa 3123 (Pi9 + Pib + Pita) with agronomy, grain and cooking quality parameters at par with PB1509. Cultivation of such improved varieties will help farmers reduce the cost of cultivation with decreased pesticide use and improve productivity with ensured safety to consumers.

Background The nucleotide-binding site–leucine-rich repeat (NBLRR) genes significantly regulate defences against various phytopathogens. The genome-wide identification and analysis of NBLRR genes have been performed in several species. However, the evolution, structure, expression of NBLRRs and functional response to Magnaporthe grisea are not known in Finger millet (Eleusine coracana L.). Results The genome-wide scanning of the Finger millet genome resulted in 116 NBLRR genes named EcNBLRRsaccording to their chromosomal positions in ascending order. The phylogenetic analysis revealed divergence of NBLRRs as per the speciation of target species. The whole-genome duplications shaped the expansion of the NBLRR family in the finger millet genome, and purifying selection (Ka/KsEcNBLRR paralog and ortholog pairs. The maximum orthology and syntenic association of NBLRRs was observed between Finger millet and Brachypodium distachyon over Finger millet and Arabidopsis thaliana. The promoter sequence analysis showed various stress- and phytohormone-responsive cis-acting elements besides growth and development, indicating their role in disease defence and regulatory mechanisms. The functional analysis of 22 EcNBLRRsin the genotypes showing contrasting responses to Magnaporthe grisea infection revealed the role of target EcNBLRRs in finger millet- Magnaporthe grisea interactions at early (EcNBLRR83; EcNBLRR55; EcNBLRR30 and EcNBLRR32) and late (EcNBLRR76; EcNBLRR45; EcNBLRR26; EcNBLRR23 and EcNBLRR21) stage of infections. Conclusion The identification and functional analysis of EcNBLRRs showed the role of EcNBLRR genes in assigning blast resistance in finger millet. These results pave the foundation for in-depth and targeted functional analysis of EcNBLRRs through genome editing and transgenic approaches.