Identification and characterization of pathogenicity-related genes of Rhizoctonia solani AG3 during tobacco infection

Abstract

Tobacco target spot disease is caused by a ubiquitous soil-borne phytopathogen Rhizoctonia solani; the pathogenic mechanisms underlying the effects of R. solani remain unclear. Deeper understanding of the functional responses to R. solani during host plant infection would help identify the molecular mechanisms essential for successful host invasion. In this study, we performed global transcriptional analysis of R. solani during various stages (12, 24, 48, 72, 96, and 120 h) of tobacco infection via an RNA sequencing method, while utilizing the pathosystem model R. solani AG3-tobacco (Nicotiana tabacum L.). After R. solani inoculation, the number of differentially expressed genes of R. solani differed at the various time points. Moreover, several gene ontology and Kyoto encyclopedia of genes and genomes pathways were unique in different infection stages, especially with respect to the genes involved in plant cell wall degradation and catalysis of biotransformation reactions, such as the pectin metabolic process and pectin catabolic process. The overexpressing-PD8 N. benthamiana plants enhanced the susceptibility to R. solani. In addition, we found that large amounts of reactive oxygen species (ROS) were generated in tobacco after infected by R. solani. R. solani encoding FAD/NAD binding oxidoreductase and peroxidase gene family to eliminating ROS and counteract oxidative stress. Moreover, Perox3 was validated that can enhance the ability of scavenging ROS by co-injecting. Overall, our findings show that pectin-degrading enzymes and cytochrome P450 genes are critical for plant infection. These results provide comprehensive insights into R. solani AG3 transcriptome responses during tobacco invasion.

Keywords: P450 genes; pectin; rhizoctonia solani; target spot disease; tobacco.

Figure 1

Disease symptoms of tobacco plants after Rhizoctonia solani inoculation.

Figure 2

Number (A) and Venn diagram (B) of total DEGs at 12, 24, 48, 72, 96, and 120 hpi.

Figure 3

GO term distribution (A) and KEGG pathway (B) in relation to R. solani–tobacco interaction.

Figure 4

Heat maps showing the expression of cytochrome P450 (A) and pectin-degrading enzymes in R. solani (B). The values shown with the bars at the right indicate the FPKM of PD genes at various time points.

Figure 5

Over-expressing PD8 lead to enhanced susceptibility to R. solani. (A) The disease phenotypes (B) The lesion diameter of leaves infected by R. solani at 2 dpi.

Figure 6

ROS production and scavenging. (A) Accumulation of ROS of tobacco plants after R. solani inoculation at 72 hpi. CK indicates non-inoculated plants. The yellow regions represent the the accumulated of ROS. (B) The expression of FNBO and Perox genes in R. solani. X-axis: 12–120 h: R. solani–inoculated tobacco harvested at various time points. Y-axis: Relative expression levels of select genes. (C) Analysis of ROS scavenging mediated by Perox3. pYBA1143: empty vector, pYBA1143-Perox3 and BAX: apoptosis regulator. The yellow regions represent the accumulated of ROS.

Figure 7

Phylogenetic tree and interaction network of pectin degrading enzymes genes. (A) The phylogenetic tree was generated from PD amino sequences using RAxML software by the Maximum Likelihood method with 500 bootstrap replicates. (B) Nodes represent proteins, and edges indicate protein-protein interaction. Edges colors indicate the type of evidence for interaction. Asterisk (*) represents significant difference (P < 0.05).

Figure 8

qPCR expression analyses of select genes at various time points post inoculation. X-axis: 12–120 h: R. solani–inoculated tobacco harvested at various time points. Y-axis: Relative expression levels of select genes in R. solani grown in PDA. Error bars indicate the mean of three biological replicates. Asterisk (*) represents significant difference (P < 0.05).