The whitefly-transmitted geminiviruses induce severe developmental abnormalities in plants. Geminivirus-encoded C4 protein functions as one of viral symptom determinants that could induce abnormal cell division. However, the molecular mechanism by which C4 contributes to cell division induction remains unclear. Here we report that tomato leaf curl Yunnan virus (TLCYnV) C4 interacts with a glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinase, designed NbSKη, in Nicotiana benthamiana. Pro32, Asn34 and Thr35 of TLCYnV C4 are critical for its interaction with NbSKη and required for C4-induced typical symptoms. Interestingly, TLCYnV C4 directs NbSKη to the membrane and reduces the nuclear-accumulation of NbSKη. The relocalization of NbSKη impairs phosphorylation dependent degradation on its substrate-Cyclin D1.1 (NbCycD1;1), thereby increasing the accumulation level of NbCycD1;1 and inducing the cell division. Moreover, NbSKη-RNAi, 35S::NbCycD1;1 transgenic N. benthamiana plants have the similar phenotype as 35S::C4 transgenic N. benthamiana plants on callus-like tissue formation resulted from abnormal cell division induction. Thus, this study provides new insights into mechanism of how a viral protein hijacks NbSKη to induce abnormal cell division in plants.

Many geminivirus C4 proteins induce severe developmental abnormalities in plants. We previously demonstrated that Tomato leaf curl Yunnan virus (TLCYnV) C4 induces plant developmental abnormalities at least partically by decreasing the accumulation of NbSKh, an ortholog of Arabidopsis BIN2 kinase involved in the brassinosteroid signaling pathway, in the nucleus through directing it to the plasma membrane. However, the molecular mechanism by which the membrane-associated C4 modifies the localization of NbSKh in the host cell remains unclear. Here, we show that TLCYnV C4 is a nucleocytoplasmic shuttle protein, and that C4 shuttling is accompanied by nuclear export of NbSKh. TLCYnV C4 is phosphorylated by NbSKh in the nucleus, which promotes myristoylation of the viral protein. Myristoylation of phosphorylated C4 favors its interaction with exportin-a (XPO I), which in turn facilitates nuclear export of the C4/NbSKh complex. Supporting this model, chemical inhibition of N-myristoyltransferases or exportin-a enhanced nuclear retention of C4, and mutations of the putative phosphorylation or myristoylation sites in C4 resulted in increased nuclear retention of C4 and thus decreased severity of C4-induced developmental abnormalities. The impact of C4 on development is also lessened when a nuclear localization signal or a nuclear export signal is added to its C-terminus, restricting it to a specific cellular niche and therefore impairing nucleocytoplasmic shuttling. Taken together, our results suggest that nucleocytoplasmic shuttling of TLCYnV C4, enabled by phosphorylation by NbSKh, myristoylation, and interaction with exportin-a, is critical for its function as a pathogenicity factor.

Summary Tomato leaf curl Yunnan virus (TLCYnV)‐encoded C4 protein induces the upregulation of the hypersensitive induced reaction 1 (HIR1) gene but interferes with the HIR1‐mediated hypersensitive response (HR). HIR1 self‐interaction is essential for the HIR1‐induced HR. TLCYnV C4 impairs the HIR1 self‐interaction and concomitantly increases the amount of Leucine‐Rich Repeat protein 1 (LRR1), a modulator of HIR1, which binds to HIR1. LRR1 promotes the degradation of HIR1, compromising the HIR1‐mediated HR. This study provides new insights into the mechanisms employed by a viral protein to counter host resistance through the cooption of the host regulatory system.