Mimic Phosphorylation of a βC1 Protein Encoded by TYLCCNB Impairs Its Functions as a Viral Suppressor of RNA Silencing and a Symptom Determinant

Abstract

Phosphorylation of the ␤C1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNB-␤C1) by SNF1-related protein kinase 1 (SnRK1) plays a critical role in defense of host plants against geminivirus infection in Nicotiana benthamiana. However, how phosphorylation of TYLCCNB-␤C1 impacts its pathogenic functions during viral infection remains elusive. In this study, we identified two additional tyrosine residues in TYLCCNB-␤C1 that are phosphorylated by SnRK1. The effects of TYLCCNB-␤C1 phosphorylation on its functions as a viral suppressor of RNA silencing (VSR) and a symptom determinant were investigated via phosphorylation mimic mutants in N. benthamiana plants. Mutations that mimic phosphorylation of TYLCCNB-␤C1 at tyrosine 5 and tyrosine 110 attenuated disease symptoms during viral infection. The phosphorylation mimics weakened the ability of TYLCCNB-␤C1 to reverse transcriptional gene silencing and to suppress posttranscriptional gene silencing and abolished its interaction with N. benthamiana ASYMMETRIC LEAVES 1 in N. benthamiana leaves. The mimic phosphorylation of TYLCCNB-␤C1 had no impact on its protein stability, subcellular localization, or self-association. Our data establish an inhibitory effect of phosphorylation of TYLCCNB-␤C1 on its pathogenic functions as a VSR and a symptom determinant and provide a mechanistic explanation of how SnRK1 functions as a host defense factor. IMPORTANCE Tomato yellow leaf curl China virus (TYLCCNV), which causes a severe yellow leaf curl disease in China, is a monopartite geminivirus associated with the betasatellite (TYLCCNB). TYLCCNB encodes a single pathogenicity protein, ␤C1 (TYLCCNB-␤C1), which functions as both a viral suppressor of RNA silencing (VSR) and a symptom determinant. Here, we show that mimicking phosphorylation of TYLCCNB-␤C1 weakens its ability to reverse transcriptional gene silencing, to suppress posttranscriptional gene silencing, and to interact with N. benthamiana ASYMMETRIC LEAVES 1. To our knowledge, this is the first report establishing an inhibitory effect of phosphorylation of TYLCCNB-␤C1 on its pathogenic functions as both a VSR and a symptom determinant and to provide a mechanistic explanation of how SNF1-related protein kinase 1 acts as a host defense factor. These findings expand the scope of phosphorylation-mediated defense mechanisms and contribute to further understanding of plant defense mechanisms against geminiviruses.KEYWORDS TYLCCNB-␤C1, Nicotiana benthamiana, SnRK1, geminivirus, host defense factor, posttranscriptional gene silencing, protein phosphorylation, transcriptional gene silencing

Nucleocytoplasmic Shuttling of Geminivirus C4 Protein Mediated by Phosphorylation and Myristoylation Is Critical for Viral Pathogenicity

“…Post-translational modifications (PTMs) play critical roles in the regulation of biological activities, including plant-pathogen interactions (Shen et al, 2011(Shen et al, , 2016Cheng et al, 2017;Zhong et al, 2017). As one of the most widespread PTMs, phosphorylation acts as an essential regulatory switch in multiple cellular processes (Manning et al, 2002a(Manning et al, , 2002bUbersax and Ferrell, 2007).…”

Herbivore-induced activation of viral phosphatase disarms plant antiviral immunities for pathogen transmission

“…We previously demonstrated that βC1 was phosphorylated by tomato SnRK1 mainly on the serine residue at position 33, and the phosphate βC1 mutant virus TA+β S33D (serine 33 to aspartate)-infected plants reduced virus accumulation and βC1 expression (Figure 2-figure supplement 3A,B) (Shen et al, 2011; Zhong et al, 2017). Serine 33 residue mutant in the βC1 protein did not affect the interactions with MPK3 and MPK6 in the subcellular localization (Figure 2-figure supplement 3C).…”

Barley stripe mosaic virus infection requires PKA‐mediated phosphorylation of γb for suppression of both RNA silencing and the host cell death response

“…VSRs are a group of viral proteins that can bind to dsRNAs (Merai et al ., ) and could be regulated by phosphorylation. A recent report on geminivirus demonstrated that phosphorylation of the viral protein βC1 destroyed its gene silencing suppression activities at both transcriptional and post‐transcriptional levels, as well as attenuating its symptom‐determinant functions (Zhong et al ., ). Deletion of a putative phosphorylation sequence or the mutation of two serine sites within this sequence in CMV 2b greatly weakened CMV‐induced symptoms (Lucy et al ., ).…”

“…Viral suppressors of RNA silencing (VSRs), including potyvirus HC‐pro (Urcuqui‐Inchima et al ., ; Valli et al ., ), Cucumber mosaic virus (CMV) 2b (Lewsey et al ., ), geminivirus βC1 (Cui et al ., ; Yang et al ., ; Zhang et al ., ; Zhong et al ., ), Barley strip mosaic virus (BSMV) γb (Yelina et al ., ; Bragg et al ., ; K. Zhang et al ., ), and Cauliflower mosaic virus (CaMV) P6 (Love et al ., ; Schoelz et al ., ), often have multiple functions in addition to RNA silencing suppression. A common and intriguing feature of viral proteins is their involvement in disease symptom development.…”

“…Phosphorylation of viral movement proteins was then shown to be a common strategy that enables plant viruses to spread (Modena et al ., ; Link et al ., ; Samuilova et al ., ; Hu et al ., ). Various viral proteins with distinct functions are phosphorylated during viral infection, including RNA‐dependent RNA polymerase (RdRp) (Kim et al ., ; Jakubiec et al ., ; Jakubiec & Jupin, ), coat protein (CP) (Ivanov et al ., ; Hung et al ., ; Zhao et al ., ), and proteins that suppress RNA silencing and symptom development (Lewsey et al ., ; Shen et al ., , ; Zhong et al ., ).…”