Identification and expression analysis of pathogenicity-related genes of Rhizoctonia solani anastomosis groups infecting rice

Abstract

Sheath blight disease caused by Rhizoctonia solani Kuhn (teleomorph; Thanatephorus cucumeris) is a major constraint in rice production. Among the different anastomosis groups (AGs) of Rhizoctonia solani, AG1-IA causes sheath blight of rice, which induce necrotic lesions on leaf sheaths of the infected plants. Several reports contradict the host specificity of anastomosis groups in Rhizoctonia solani. There is lack of information on the pathogenicity genes of these Rhizoctonia solani anastomosis groups during sheath blight infection in rice. In the present study, Rhizoctonia solani isolates collected from diverse rice growing regions of India were screened for anastomosis groups and two groups namely, AG1-IA, AG2-2 were identified. Accordingly, comparative studies were made with AG1-IA (GenBank ID: 16,395) and AG2-2 (GenBank ID: 2,318,768) group sequences, which enabled the identification of specific gene clusters (119 in AG1-IA and 604 in AG2-2) belonging to these groups. Pathogen Host Interaction (PHI) blast with these specific gene clusters could further identify genes involved in host pathogen interaction (38 in AG1_IA and 150 in AG2-2), which were shortlisted for qRT-PCR validation based on qcov cutoff values representing different phenotypic categories of PHI blast. Expression analysis-based validation in sheath blight susceptible (Pusa Basmati 1) and resistant (Pusa 1908-13-12-5) rice genotypes showed that most of the genes expressed significantly higher in the susceptible variety Pusa Basmati 1. The genes like inorganic phosphate transporter (AG1_IPT), Bromodomain containing protein (AG1_BrD), Aldehyde dehydrogenase (AG1_AldD), AMP binding domain (AG1_AMP) and Heme peroxidase (AG1_HmPr) were upregulated in the susceptible genotype, PB 1 at 72hpi compared to Pusa 1908-13-12-5. Among these, inorganic phosphate transporter (AG1_IPT), Bromodomain containing protein (AG1_BrD) and Heme peroxidase (AG1_HmPr) were specific to Rhizoctonia solani AG1-IA. Through the present study, we could demonstrate the AG1-IA-specific interactions of Rhizoctonia solani causing sheath blight disease of rice, which is a step forward in understanding the specificity of Rhizoctonia solani with reference to sheath blight disease of rice.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-021-02934-1.

Keywords: AG1-IA; AG2-2; Inorganic phosphate transporter; PB 1; Sheath blight.

Fig. 1

Rhizoctonia solani anastomosis group isolates. a AG1-IA: UP114, b AG2-2: UP105

Fig. 2

Pathogenicity test in susceptible and resistant rice genotype. Relative lesion height was observed highest in susceptible variety Pusa Basmati 1 as compared to tolerant genotype Pusa 1908-13-12-5

Fig. 3

Phylogenetic tree showing the clusters of Rhizoctonia solani isolates. UP114 in AG1-IA group and UP105 in AG2-2. AG5 ND2 taken as outgroup. ClustalW-based alignment was used to construct the tree based on Neighbor Joining method with bootstrap value of 1000 replicates

Fig. 4

Unique, orthologous and size of gene clusters in Rhizoctonia solani AG1-IA and AG2-2 isolates

Fig. 5

Functional annotation of genes in Rhizoctonia solani AG1-IA and AG2-2. Top ten hits of biological process, molecular function and cellular component were annotated. a Rhizoctonia solani AG1-IA, b Rhizoctonia solani AG2-2

Fig. 6

Pathogen–host interactions (PHI-base) database phenotypic categories of Rhizoctonia solani AG1-IA and AG2-2

Fig. 7

PCR analysis of Rhizoctonia solani 18S rDNA and pathogenicity genes. a rice cDNA samples amplified with pathogen specific 18S rDNA: M = Molecular marker:100 bp, Lane 1–3: PB1 inoculated with UP114 (AG1-IA) at 24, 48 and 72 h post inoculation, respectively, Lane 4–6: Pusa 1908–13-12–5 inoculated with UP114 (AG1-IA) at 24, 48 and 72 h post inoculation, respectively, Lane 7–9: PB1 inoculated with UP105 (AG2-2) at 24, 48 and 72 h post inoculation, respectively, Lane 10–12: Pusa 1908–1 inoculated with UP105 (AG2-2) at 24, 48 and 72 h post inoculation, respectively. b Pathogenicity genes found specific to Rhizoctonia solani AG1-IA: M = Molecular marker:100 bp, Lane 1–4: Gene inorganic phosphate transporter with DNA of AG1 (UP114), AG2 (UP105), AG3 (RPBC 1), AG5 (RMHM 6), respectively, Lane 5–8: Bromodomain containing protein with DNA of AG1, AG2, AG3, AG5 respectively, Lane 9–12: Gene Heme peroxidase with DNA of AG1, AG2, AG3 and AG5, respectively. c Pathogenicity gene found specific to Rhizoctonia solani AG2-2: M = Molecular marker:100 bp, Lane 1–4: Gene Lissencephaly-1 homolog with AG1, AG2, AG3, AG5, respectively. d Pathogenicity genes selected from previous studies (Rioux et al. 2011): M: Molecular marker 100 bp; lane 1–2: gene GLU with AG1,AG2; lane 5–6: gene AAA with AG1, AG2; lane 9–10: gene NIC with AG1, AG2

Fig. 8

qRT PCR expression analysis of Rhizoctonia solani genes in susceptible (PB1) and resistant (Pusa 1908-13-12-5) genotypes at different hours of post inoculation. a Inorganic phosphate transporter (AG1_IPT), b Bromodomain function (AG1_BrD), c Aldehyde dehydrogenase (AG1_AldD), d AMP binding domain (AG1_AMP), e Heme peroxidase (AG1_HmPr), f Lissencephaly-1 homolog (AG2_LisH), g Ephrin type-A receptor (AG2_HyMt), h Glutathione-s-transferase kappa 1 (RS_GLU) expression pattern by AG1-IA, i Glutathione-s-transferase kappa 1 (RS_GLU) expression pattern by AG2-2, j Nuclear pore protein, Nic96 (RS_NIC) induced by AG1-IA, k Nuclear pore protein, Nic96 (RS_NIC) in AG2-2. a–e Expression pattern of Rhizoctonia solani_AG1-IA pathogenicity genes, a, b and e: Expression pattern of AG1-IA-specific pathogenicity genes of Rhizoctonia solani. f–g Expression pattern of Rhizoctonia solani_AG2-2 pathogenicity genes. h–k Expression pattern of Rhizoctonia solani pathogenicity genes which were previously studied in rice and potato pathosystems by Rioux and coworkers, 2011