Whole-genome sequence analysis of Bipolaris sorokiniana infecting wheat in India and characterization of ToxA gene in different isolates as pathogenicity determinants

Abstract

Spot blotch disease of wheat caused by Bipolaris sorokiniana Boerma (Sacc.) is an emerging problem in South Asian countries. Whole genome of a highly virulent isolate of B. sorokiniana BS112 (BHU, Uttar Pradesh; Accession no. GCA_004329375.1) was sequenced using a hybrid assembly approach. Secreted proteins, virulence gene(s), pathogenicity-related gene(s) were identified and the role of ToxA gene present in this genome, in the development of disease was recognized. ToxA gene (535 bp) was analyzed and identified in the genome of B. sorokiniana (BS112) which revealed 100% homology with the ToxA gene of Pyrenophora tritici repentis (Accession no. MH017419). Furthermore, ToxA gene was amplified, sequenced and validated in 39 isolates of B. sorokiniana which confirmed the presence of ToxA gene in all the isolates taken for this study. All ToxA sequences were submitted in NCBI database (MN601358-MN601396). As ToxA gene interacts with Tsn1 gene of host, 13 wheat genotypes were evaluated out of which 5 genotypes (38.4%) were found to be Tsn1 positive with more severe necrotic lesions compared to Tsn1-negative wheat genotypes. In vitro expression analysis of ToxA gene in the pathogen B. sorokiniana using qPCR revealed maximum upregulation (14.67 fold) at 1st day after inoculation (DAI) in the medium. Furthermore, in planta expression analysis of ToxA gene in Tsn1-positive and Tsn1-negative genotypes, revealed maximum expression (7.89-fold) in Tsn1-positive genotype, Agra local at 5th DAI compared to Tsn1-negative genotype Chiriya 7 showing minimum expression (0.048-fold) at 5th DAI. In planta ToxA-Tsn1 interaction studies suggested that spot blotch disease is more severe in Tsn1-positive genotypes, which will be helpful in better understanding and management of spot blotch disease of wheat.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-022-03213-3.

Keywords: Bipolaris sorokiniana; Spot blotch; ToxA; Tsn1; Whole-genome sequence; qPCR.

Fig. 1

Top 10 PFam domain distribution in the whole genome of B. sorokiniana using HMMER 3.0

Fig. 2

Top 10 genes expressing the type of virulence/pathogenicity from whole-genome functional annotation of B. sorokiniana using pathogen–host interaction (PHI) database

Fig. 3

Pie chart representing gene ontology based functional annotation of genes present in the B. sorokiniana genome. GO charts were generated using. a Top 15 process hits for gene ontology biological process (3300). b Top 15 process hits for gene ontology molecular function (7784). c Top 15 process hits for gene ontology cellular component (4165)

Fig. 4

a Pairwise genome alignment using Dashing tool among seven genomes of B. sorokiniana isolates (BS112.fna, BRIP10943a.fna, WAI2406.fna, WAI2411.fna, BRIP27492a.fna, ND90Pr.fna, Shoemaker.fna) and one isolate of P. tritici-repentis (AR_CrossB10.fna, outgroup). Phylogenetic tree was created using MEGA X. b Venn diagram showing unique and shared orthologous gene families among four Bipolaris species namely Cochliobolus victoriae/ B. victoriae, Accession no. GCA_000527765, Strain- F13; C. miyabeanus/B. oryzae Accession no. GCA_000523455.1, Strain- ATCC 44,560; C. sativus/B. sorokiniana, BS112; and C. carbonum/B. zeicola Accession no. GCA_000523435.1, Strain- 26-R-13

Fig. 5

Functional annotation of the B. sorokiniana secretome showing topmost category Viz. MF molecular function (348), CC cellular component (88), BP biological process (137)

Fig. 6

CAZymes class annotation distribution in the genome of B. sorokiniana BS112. All six CAZyme categories are represented as: AA Auxiliary activities (10), CBM Carbohydrate binding modules (18), CE Carbohydrate esterase (6), GH Glycoside hydrolases (45), GT Glycosyl transferases (1), and PL Polysaccharide lyases (4)

Fig. 7

a Amplification profile of wheat genotypes harboring Tsn1 gene (400 bp amplicon). M (100 bp bioprep marker), 1-WH 542, 2-WL 711, 3-Agra Local, 4-Suzoe, 5- HD 29, 6- PBW 343, 7-HD 3086, 8-Chiriya 7, 9-HD 30, 10-Milan, 11-HD 2329, 12- Ning 8139, 13-HD 2967. b Average Disease Index (ADI) on 3-week-old seedlings of different wheat genotypes (1-WH 542, 2-WL 711, 3-Agra Local, 4-Suzoe, 5- HD 29, 6- PBW 343, 7-HD 3086, 8-Chiriya 7, 9-HD 30, 10-Milan, 11-HD 2329, 12- Ning 8139, 13-HD 2967) at 4 days post-inoculation with virulent isolate BS112 of B. sorokiniana

Fig. 8

a In vitro expression analysis of ToxA gene in B. sorokiniana pathogen collected at different time intervals (1st, 2nd, 3rd, 4th and 5th day) in liquid broth medium. Error bars shows ± SD among the biological triplicates. b In vivo expression analysis of ToxA gene after inoculation on two different wheat genotypes at different time points (1st, 2nd, 3rd, 4th and 5th day). Error bars shows ± SD among the biological triplicates