Leaf spot is a new type of fungal disease that restricts the production of broad beans, and Alternaria alternata is one of its main pathogens. The objective of this study is to explore the molecular pathways of broad beans in response to the infection of A. alternata through transcriptome analysis and disease resistant gene function verification.

Broad bean cultivar CD-006 was used as the experimental material, transcriptome sequencing was performed on the leaves inoculated with A. alternata at 0, 6, 12 and 24 h to screen differentially expressed genes (DEGs). GO (gene ontology) functional annotation and KEGG (Kyoto encyclopedia of genes and genomes) metabolic pathway enrichment analysis were conducted to excavate disease resistance related genes. qRT-PCR (quantitative real-time PCR) was conducted to validate transcriptome sequencing results, and VfPR4 was screened for genetic transformation in tobacco. To complete disease resistance function analysis of VfPR4, invasive inoculation method was used.

At 6, 12 and 24 h after infection, 3 537, 3 152 and 2 947 DEGs were up-regulated in broad beans, while 1 181, 1 453 and 1 319 DEGs were down-regulated. GO enrichment analysis revealed that DEGs were mainly enriched in oxidoreductase activity and some biosynthetic (metabolic) processes, and KEGG enrichment analysis found that DEGs were mainly enriched in pathways such as biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and flavonoid biosynthesis. At 6 h after infection, KEGG was also enriched in MAPK (mitogen-activated protein kinase) signaling pathway and plant-pathogen interaction pathway, which together enriched 32 DEGs. A total of 52 PR genes were discovered in transcriptome analysis, among which 35 genes were differentially expressed. Six DEGs were selected for qRT-PCR validation, and the validation results were consistent with the transcriptome sequencing results. Among the above genes, the VfPR4 remained up-regulated within 24 h after infection with A. alternata. Tobacco genetic transformation results indicated that the lesion symptoms of transgenic lines were milder, with lesion areas of 128.94, 110.57 and 92.92 mm², respectively, which were significantly smaller than that of the wild type, with a lesion area of 174.32 mm².

In this study, transcriptome analysis revealed that broad beans might mainly respond to the infection of A. alternata through pathways including oxidative burst, secondary metabolites biosynthesis, phenylpropanoid biosynthesis, MAPK signaling, and plant-pathogen interaction. VfPR4 could positively regulate tobacco's resistance to A. alternata.