The role of cysteine-rich protein in enhancing mandarivirus infectivity and pathogenicity.

Mandariviruses pose a significant threat to the citrus industry due to their diverse transmission modes and high pathogenicity. However, the pathogenicity mechanisms of mandariviruses remain largely unknown, especially as there is a complete lack of understanding regarding the function of the cysteine-rich proteins (CRPs) encoded by mandariviruses during infection. In this study, ectopic expression of two mandarivirus CRPs from citrus yellow mottle-associated virus (CiYMaV) and citrus yellow vein clearing virus (CYVCV) using a potato virus X vector resulted in severe symptoms and increased viral accumulation in Nicotiana benthamiana. CiYMaV CRP induced cell death in N. benthamiana leaves, with the zinc finger motif identified as the critical region responsible for this induction. CYVCV CRP functioned as a suppressor to inhibit local gene silencing induced by single-stranded GFP, but not double-stranded GFP. Furthermore, mutational analysis of the infectious clones of CiYMaV and CYVCV revealed that their respective CRPs are essential for disease symptom development and viral accumulation in citrus plants. In summary, our findings indicate that CiYMaV and CYVCV CRPs act as pathogenicity determinants, thereby enhancing our understanding of the functional repertoire within the mandariviruses proteome and providing a target for citrus hosts to defend against mandariviruses.

Importance: Mandariviruses, infecting a wide range of citrus varieties, cause serious epidemics in Pakistan, India, Turkey, China, Iran, Italy, and America. However, little information is available about pathogenicity mechanisms of mandariviruses. Here, we confirmed the importance of two mandarivirus CRPs of citrus yellow mottle-associated virus (CiYMaV) and citrus yellow vein clearing virus (CYVCV) in disease symptom development and viral accumulation in citrus plants. Our study first provides evidence that CiYMaV and CYVCV CRPs, nonstructural proteins, act as pathogenicity determinants with multiple functions. This offers a broad understanding of functional repertoire within the mandariviruses proteome. Further investigation of the underlying mechanisms of how CRP, as a virulence factor, modulates plant immunity may suggest a possible new strategy for combating mandarivirus infection in the field.