5. CROP PROTECTION
5.1.2 Pathogenicity, host-pathogen interaction and genomics
Genome-based identification of simple sequence repeats markers in Tilletia indica population: A total of 5,772 simple sequence repeat loci were identified in T. indica genome (MBSW00000000). Maximum SSRs were tri-nucleotide (2,456) followed by di-nucleotides in the genome. In silico analysis, 40 microsatellite markers were used to genotype 20 Tilletia indica isolates belonged to north-western plain zone of India. In total, 130 alleles were identified. The highest number of alleles were 6 in TISSR1 and TISSR34 markers. The
(a) Little leaf and witches’ broom disease in peanut and (b) Phylogenetic tree constructed by the neighbor-joining method of 16S rRNA gene of the peanut
a b
frequency of 2 loci was very high in TI population. The polymorphic information value content (PIC) values ranged from 0.20 to 0.81 with an average of 0.51. The highest PIC value was 0.81 in TISSR34 followed by TISSR1 (0.75).
Transcriptome analysis of resistant and susceptible genotypes infected with Bipolaris sorokiniana causing spot blotch of wheat: Whole transcriptome analysis was performed on B. sorokiniana-wheat interaction.
Transcriptome of B. sorokiniana inoculated resistant (Chiriya 7) and susceptible (Agra Local) genotypes of wheat were analyzed. Approximately 33 million reads were generated using RNA sequencing by Illumina NextSeq500 platform. An average of 81.20%
of the reads was aligned to the reference genome.
Differential expression was performed to identify differentially regulated gene in two genotypes. Out of 2938 genes, 1408 were significantly up-regulated whereas 1530 genes were significantly down-regulated on the basis of p-values in Chiriya 7 inoculated with B.
sorokiniana. Out of 2,293 genes, 1,005 were significantly up-regulated, whereas 1,288 genes were significantly down-regulated on basis of p-values in Agra local inoculated with B. sorokiniana. Transcripts of Chiriya 7 and Agra local inoculated with B. sorokiniana were also compared to B. sorokiniana transcripts. Out of 24 genes, 18 were significantly up-regulated whereas 6 genes were significantly down-regulated in Chiriya 7. Out of 32 genes, 16 were significantly up-regulated whereas 16 genes were significantly down-regulated in Agra local.
Transcriptomic analysis of bakanae disease resistant and susceptible rice genotypes in response to infection by Fusarium fujikuroi: Two contrasting genotypes of rice i.e. resistant (C101A51) and susceptible (Rasi) to bakanae disease were taken and transcriptome analysis was performed. Transcriptomic analysis was conducted between C101A51 control (CC) vs C101A51 inoculated (CI), Rasi control (RC) vs Rasi inoculated (RI) and C101A51 inoculated (CI) vs Rasi inoculated (RI). In CC vs CI, differentially expressed genes (DEGs) were 12,764 (79%). Out of them, 567 (4%)
were significantly upregulated and 1399 (9%) genes were down regulated. For the RC vs RI 14, 333 (79%) genes were commonly expressed in both inoculated plants and control plants. When we compared CI vs RI ,13,662 (72%) genes were identified to be commonly expressed. Further, 280 (1%) genes were exclusive in CI and 532 (3%) genes were exclusive in RI. Cysteine proteinase inhibitor 10, disease resistance protein TAO1-like, oleosin 16 kDa-like, pathogenesis-related protein (PR1), pathogenesis-related protein (PR4), BTB/POZ and MATH domain-containing protein 5-like, alpha-amylase isozyme 3D-like (LOC4345814), were upregulated in resistant genotype C101A51.
Whereas, GDSL esterase/lipase At5g33370, serine glyoxylate aminotransferase, CASP-like protein 2C1, WAT1-related protein At4g08290, cytoplasmic linker associated proteins, xyloglucan endotransglucosylase/
hydrolase protein and β-D xylosidase 7 were found to be upregulated in susceptible genotype Rasi. This study will be helpful for the development of bakanae resistant rice varieties.
Phytotoxin as a key component in pathogenicity and virulence of Rhizoctonia solani inciting sheath blight of rice: In the process of identifying interacting partners in rice for potential pathogenicity factors of Rhizoctonia solani inciting sheath blight, the role of host-selective toxins elaborated by the fungal pathogen in ShB development has been elucidated. The fungal isolates were collected from six locations and preserved as laboratory collections. These were characterized using their cultural and morphological characteristics and their identities were confirmed by PCR amplification of ITS region and sequencing. Pathogenicity assay was established in the polyhouse under artificial inoculation conditions. The phytotoxin was then extracted from the highly virulent isolate using ethyl acetate and tested for lesion development by both detached leaf and in- planta assays. The phytotoxin of the R. solani isolates was observed to reproduce symptoms of the disease independent of the pathogen itself, and induced maximum lesion development for a highly pathogenic isolate RIRS-K confirming its role as a key component in pathogenicity of the necrotrophic fungus.
Effect of crude RS-Toxin on ShB development on 60 days old rice cv PB-1 at various concentrations (A) in planta assay (i)10,000 ppm (Stock) (ii)1000 ppm (iii) 500 ppm (iv) aqueous fraction (v) acetone (vi) water (vii) RIRS-K
Virulence analysis of powdery mildew (Blumeria graminis tritici) isolates: The powdery mildew infected wheat disease samples collected from different places viz., Rampur, Pantnagar, Haldwani, Dhaulakuan, Bilaspur, Katrain, Shimla, Gurdaspur, Ludhiana, Pathankot and Jammu were used for virulence analysis using host differential sets consisting 14 near- isogenic powdery mildew lines each carrying single Pm-gene along with two universal susceptible checks.
Avirulence and virulence formulae of Bgt culture were also used to known the effective and ineffective genes.
Virulence was observed on gene Pm3a, Pm3b, Pm3c, Pm5, Pm7 and Pm8. No virulence was detected on gene Pm1, Pm2, Pm4a and Pm6.
Identification and expression analysis of a virulence gene Tox A in Bipolaris sorokiniana causing spot blotch of wheat: Tox A gene has been identified and characterized in Indian isolates of Bipolaris sorokiniana.
An amplicon of 600 bp (approx.) was sequenced. The analysis revealed 100% homology with Tox A gene in Pyrenophora tritici repentis. All these BsToxA sequences have been submitted in NCBI database (MN601358- MN601396). In vitro expression analysis of ToxA gene in B. sorokiniana isolate (BS112) using qPCR revealed maximum up-regulation (14.67) at 1st day after
inoculation (DAI), followed by 2nd DAI (11.83 fold), then gradually expression level decreased at 3, 4 and 5 DAI. In planta, expression analysis was also performed using qPCR. The maximum expression (2.13-fold) was observed at 3rd DAI in susceptible cultivar (Agra local), while minimum expression (0.23-fold) was observed in resistant genotype (Chiriya 7) at 3rd DAI .
Comparative genomics of Magnaporthe species infecting rice and pearlmillet: Whole genomes of Magnaporthe oryzae strain RMg-Dl infecting rice and M. grisea strain PMg-Dl infecting pearl millet was sequenced using the Illumina and PacBio platforms.
The high throughput hybrid assembly of short and long reads resulted to 341 and 996 scaffolds with the genome size of 47.89 Mb and 34.81 Mb of PMg-Dl and RMg-Dl strain, respectively. Comparative genome analysis revealed 83% average nucleotide identity (ANI) and gene calling confirmed a total of 10451 and 12,747 genes in both genomes with common and unique proteins/genes, protein family and superfamily between the genomes. A total of 5,589 and 5,032 genes were uniquely present in both genomes. Similarly, the Inter Pro Scan of predicted protein sequences of both genomes annotation revealed that 148 (4.3%) and 65 (1.9%) protein family (PFAM), 38 (7.7%) and 29 (5.9%) super family (PIRSF) were uniquely present in PMg-Dl and RMg-Dl genome. The prevalence of Virulence Factors (VF) determination revealed that majority (90.9%) of VFs were shared between both the genomes. However, two VFs were unique in PMg-Dl and three VFs were unique in case of RMg- Dl genome. Comparative annotation of whole genome of M. grisea and M. oryzae infecting pearl millet and rice revealed that genome size was larger in pearl millet infecting M. grisea than rice infecting M. oryzae indicating genome reduction in rice adapted M. oryzae.
Comparative genome analysis also revealed specific virulence factors, CAZmes and unique pathways in pearl millet infected by M. griseawhen compared to other Magnaporthe strains infecting rice.
Comparative genome analysis of Tilletia indica reveals high genomic variation: The whole genome of Tilletia indica RAKB_UP_1 have been sequenced
(GCA_002220835.1). The genomes have variable size ranging from 26.7 Mb to 43.7 Mb. The genomes of seven isolates of T. indica were compared. For genome mapping, all the genome assemblies were aligned against T. indica RAKB_UP_1 genome using minimap2.
Tik_1 was mapped 100% with the reference genome.
Further, comparative genome analysis was done by identification of SNPs, In-Dels, segmental duplications, LTR and CNV. It revealed that variable number of SNPs were ranging from 1380 (Tik_1) to 160966 (Tik) and 82 (PSWKBGH_1) to 3309 (PSWKBGD_1_3) In- Dels. Copy number variations including duplications and deletions were also identified, while Tik had shown highest number of duplicate counts (100).
Agrobacterium-mediated in planta transformation of cotton with antisense βC1 gene construct (pCAMBIA-As-βC1): Agrobacterium-mediated in planta transformation with antisense βC1 gene construct (pCAMBIA-As-βC1) was carried out in 5 cotton vars.
Coker 310, RST-9, F846, HS6 and PSS-2. The GUS assay and PCR analysis of T1 cotton plants showed that two cotton vars. Coker 310 and RST-9 were transformed with pCAMBIA-As-βC1 gene construct. Stable segregation of As-βC1 transgene in both the cotton varieties was evaluated upto T2 generation. RT-PCR of T2 cotton plants confirmed βC1 and nptII gene expressing in T2 transgenic line. T3 cotton plants will be challenged with viruliferous whitefly to check the resistance capacity against CLCuD begomovirus.
Development of mutagenic construct of avrBs1 in suicide vector (pK18mobSacB): The mutagenic primers of avrBs1 having restriction enzyme site. i.e. EcoRI and XbaI in fragment 1 (F1) and XbaI and EcoRI in fragment 2 (F2) of avrBs1 gene were amplified by proof reading Taq polymerase. The amplified products of F1 and F2 fragments were digested with XbaI restriction enzyme and ligated by DNA ligase. The ligated product was cloned in pGEMT-Easy vector. The M13 forward and reverse primers were used to confirm the cloned product by Colony PCR. The clone of F1F2/pGEMT is using for the generation of mutagenic construct of avrBs1 in pk18mobsacB, suicide vector. Additionally, the mutagenic construct of avrBs1 gene of Xcc was
developed and checked by colony PCR and restriction digestion analysis. The developed construct was confirmed by DNA sequencing.
Role of suppressor proteins of begomovirus in viral pathogenicity: Three putative silencing suppressor proteins (V2, C2, and C4) of croton yellow vein mosaic virus (CYVMV) were amplified and cloned in pEarleyGate103 vector. Transient expression of each of V2, C2 and C4 proteins in Nicotiana benthamiana resulted in typical leaf curl symptom in systemic leaves. Further analysis through RT-PCR and confocal microscopy showed the expression of these proteins in systemic leaves, indicating that all of them act as pathogenicity determinants.
Development of hairpin-RNAi constructs against the suppressor genes of tomato leaf curl New Delhi virus and their evaluation through transient expression:
Three suppressor genes (AV2, AC2 and AC4) of tomato leaf curl New Delhi (ToLCNDV) was amplified and hairpin RNAi constructs against them was developed using a high throughput golden gate based binary vector pRNAiGG. The efficiency of agro-constructs was evaluated through transient expression in N.
benthamiana followed by challenged inoculation of ToLCNDV. qPCR analysis at 4 dpi indicated that the virus accumulation in the inoculated plant reduced significantly in all the hairpin RNA inoculated plants.
Highest reduction was observed when the plants were treated with AC4 hairpin RNA.
Understanding the minimum size of begomovirus replication module to be used as vector: To understand the minimum viral replicon size of croton yellow vein mosaic virus (CYVMV), six constructs (gene deleted and point mutation) were developed. To understand the replication behavior of these constructs and the symptom phenotype produced by them, all the constructs were agroinfiltrated into N. benthamiana plants. None of the constructs developed any symptom phenotype even after 30 dpi. Further molecular analysis for understanding the replication of these molecules indicated that they are efficiently able to replicate in the inoculated area but could not move systemically.
Result thus suggested that for replication C1-C3 portion are essentially required and V2-V1 portion are essentially required for cell-to-cell and long-distance movement of the virus. V2-V1 deleted minimum viral replicon module can effectively express GFP in the inoculated tissues.