Crop vascular diseases are one of the most serious diseases affecting agricultural production and food security. They are highly harmful and difficult to control. Related pathogens, rice bacterial blight and Ralstonia solanacearum, infect the plant's vascular system, occupy and block the xylem of the vascular bundle, thereby affecting the transport of water and nutrients in the plant, and eventually causing the plant to wilt and die. Biofilm is an important strategy for bacteria to resist harsh living environments, antibacterial substances or host immune responses. However, how vascular pathogens represented by R. solanacearum regulate the dynamic changes of biofilms in the host and help bacteria colonize and spread in the vascular bundle is still unclear.
Recently, Li Bo's research group from Huazhong Agricultural University published a research paper titled "Holliday junction resolvase RuvC targets biofilm eDNA and confers plant resistance to vascular pathogens" in Nature Plants, revealing the molecular mechanism of how R. solanacearum uses the Holliday junction (HJ) resolvase RuvC to promote the dissociation of mature biofilms and bacterial spread. In addition, new tomato germplasms with broad-spectrum resistance to R. solanacearum were created by targeting biofilm formation, and the relevant disease resistance improvement strategy was also verified in rice bacterial blight. This study provides new ideas for the discovery and creation of resources for resistance to vascular diseases and provides new resources with application value for the breeding of durable disease-resistant varieties.
Through mass spectrometry analysis of the biofilm proteome of R. solanacearum GMI1000, a highly abundant Holliday junction (HJ) resolvase RuvC was identified. Knocking out RuvC in R. solanacearum affects the pathogenicity to tomatoes and inhibits the spread and migration of R. solanacearum in the xylem. Analysis found that the knockout mutant resulted in excessive accumulation of biofilm and eDNA, while the grid-like structure of eDNA was significantly enhanced in the RuvC mutant. Further research found that RuvC can be secreted outside bacterial cells and co-localized with the Holliday-like structure of biofilm eDNA. In vitro enzymatic activity experiments demonstrated that RuvC can degrade DNA with a cruciform structure, and its resolving enzyme activity is necessary for the virulence of R. solanacearum. Adding purified RuvC to the in vitro biofilm culture system can significantly inhibit the formation of R. solanacearum biofilm. By detecting the expression dynamics of R. solanacearum RuvC in vitro and in tomato xylem, it was found that RuvC expression was induced in the late stages of biofilm maturation. Based on the above research results, a dynamic regulation model of R. solanacearum biofilm was proposed. In the early stages of colonization, R. solanacearum forms biofilms to occupy ecological space and reproduce. After the organism matures and lacks nutrients and space, bacteria secrete RuvC to degrade the Holliday-like structure of eDNA, causing part of the biofilm to disintegrate and promote bacterial spread and infection.
Figure 1. RuvC regulating biofilm dynamics. (Du, et al., 2024)
Based on the degradation function of HJ structural dissociation enzyme on R. solanacearum biofilm, the research team created a series of new germplasm materials with broad-spectrum resistance to crop vascular bacterial diseases. The N-terminus of the RuvC protein of R. solanacearum was fused to the secreted signal peptide of the plant disease progression-related protein PR1, and tomatoes and tobacco expressing the secreted protein were transformed. It was found that the transgenic material improved the disease resistance of Solanaceae crops to different strains of R. solanacearum. The survival rate of tomatoes and tobacco after blight infection is increased by about 50%. In addition, the Xanthomonas oryzae homologous protein XooRuvC with a signal peptide was expressed in the Nipponbare background, and it was found that the resistance of the transgenic rice to bacterial blight was significantly improved. In addition, phylogenetic analysis revealed that MOC1, a protein with a RuvC-like functional domain, is ubiquitous in plants and is located in chloroplasts. In vitro expression and purification of tomato and rice MOC1 recombinant proteins that do not contain chloroplast transit peptides have strong inhibitory effects on the biofilms of R. solanacearum and X. oryzae. Moreover, expression of secreted SlMOC1 in tomato hairy roots significantly improved tomato resistance to R. solanacearum. The above research results show that expressing and secreting HJ structural dissociation enzymes derived from bacteria or plants into plant cells can effectively improve crop resistance to bacterial vascular diseases. Targeting bacterial biofilms is a new idea for improving crop disease resistance.
