Objective: Xanthomonas oryzae pv. oryzae (Xoo) possesses a type III secretion system (T3 S), encoded by a hpa-hrp-hrc cluster, including hrpD6, to inject T3S effectors into plant cells to trigger hypersensitive response (HR) in nonhost tobacco and pathogenicity in susceptible host rice. However, it is unclear what roles of Xoo hrpD6 gene plays in HR in tobacco and in pathogenicity in rice.
Methods: In this study, we constructed a deletion mutant of hrpD6 gene by using marker-exchange method. PCR and Southern blot analysis demonstrated that the hrpD6 gene was knocked out successfully.
Results: in planta assays indicated the hrpD6 mutant, delta PhrpD6, lost the ability to induce HR in tobacco, to trigger water-soaked symptoms in seedlings rice and to cause bacterial blight in adult rice. Importantly, the bacterial growth in rice tissues was tremendously reduced. Complementation assays confirmed that hrpD6 gene could restore HR induction in tobacco, pathogenicity and bacterial growth in rice to the mutant delta PhrpD6. Reverse transcriptional polymerase chain reaction (RT-PCR) revealed that the expression of hrpD6 was not only induced by rice cells, but also controlled by hrpG and hrpX. Intriguyingly, the expression of hpal, encoding a harpin protein, was found to be dependent on hrpD6, implying that hrpD6 regulates the expression of hpal. Immunobloting assay confirmed that the mutation of hrpD6 affect the secretion of Hpal through T3S.
Conclusion: The mutant lost the ability of triggering hypersensitive response in nonhost tobacco and pathogenicity in host rice is due to that hrpD6 regulates the expression of hpal gene and the mutation in hrpD6 affects the secretion of T3S effectors, like Hpa1, through T3SS. Our results provide molecular clues to understand whether hrpD6 is involved in the formation of T3S apparatus and in regulation of other hpa-hrp-hrc gene expression or not for HR induction in tobacco and pathogenicity in rice.