The C4 protein encoded by tomato leaf curl Yunnan virus reverses transcriptional gene silencing by interacting with NbDRM2 and impairing its DNA-binding ability.

In plants, cytosine DNA methylation is an efficient defense mechanism against geminiviruses, since methylation of the viral genome results in transcriptional gene silencing (TGS). As a counter-defense mechanism, geminiviruses encode viral proteins to suppress viral DNA methylation and TGS. However, the molecular mechanisms by which viral proteins contribute to TGS suppression remain incompletely understood. In this study, we found that the C4 protein encoded by tomato leaf curl Yunnan virus (TLCYnV) suppresses methylation of the viral genome through interacting with and impairing the DNA-binding ability of NbDRM2, a pivotal DNA methyltransferase in the methyl cycle. We show that NbDRM2 catalyzes the addition of methyl groups on specific cytosine sites of the viral genome, hence playing an important role in anti-viral defense. Underscoring the relevance of the C4-mediated suppression of NbDRM2 activity, plants infected by TLCYnV producing C4(S43A), a point mutant version of C4 unable to interact with NbDRM2, display milder symptoms and lower virus accumulation, concomitant with enhanced viral DNA methylation, than plants infected by wild-type TLCYnV. Expression of TLCYnV C4, but not of the NbDRM2-interaction compromised C4(S43A) mutant, in 16c-TGS Nicotiana benthamiana plants results in the recovery of GFP, a proxy for suppression of TGS. This study provides new insights into the molecular mechanisms by which geminiviruses suppress TGS, and uncovers a new viral strategy based on the inactivation of the methyltransferase NbDRM2.

Divergent Symptoms Caused by Geminivirus-Encoded C4 Proteins Correlate with Their Ability To Bind NbSKη.

Geminiviruses induce severe developmental abnormalities in plants. The C4/AC4 protein encoded by geminiviruses, especially those not associated with betasatellites, functions as a symptom determinant by hijacking a shaggy-related protein kinase (SKη) and interfering with its functions. Here, we report that the symptom determinant capabilities of C4 proteins encoded by different geminiviruses are divergent and tightly correlated with their abilities to interact with SKη from Nicotiana benthamiana (NbSKη). Swap of the minidomain of tomato leaf curl Yunnan virus (TLCYnV) C4 critical for the interaction with NbSKη increases the capacities of the C4 proteins encoded by tomato yellow leaf curl China virus (TYLCCNV) or tobacco curly shoot virus (TbCSV) to induce symptoms. The severity of symptoms induced by recombinant TYLCCNV C4 or TbCSV C4 correlates with the amount of NbSKη tethered to the plasma membrane by the viral protein. Moreover, a recombinant TYLCCNV harboring the minidomain of TLCYnV C4 induces more-severe symptoms than wild-type TYLCCNV. Thus, this study provides new insights into the mechanism by which different geminivirus-encoded C4 proteins possess divergent symptom determinant capabilities.IMPORTANCE Geminiviruses constitute the largest group of known plant viruses and cause devastating diseases in many economically important crops worldwide. Geminivirus-encoded C4 protein is a multifunctional protein. In this study, we found that the C4 proteins from different geminiviruses showed differential abilities to interact with NbSKη, which correlated with their symptom determinant capabilities. Moreover, a minidomain of tomato leaf curl Yunnan virus (TLCYnV) C4 that is indispensable for interacting with NbSKη and tethering it to the plasma membrane, thus leading to symptom induction, was determined. Supporting these findings, a recombinant geminivirus carrying the minidomain of TLCYnV C4 induced more-severe symptoms than the wild type. Therefore, these findings expand the scope of the interaction of NbSKη and C4-mediated symptom induction and thus contribute to further understanding of the multiple roles of C4.

The C4 protein encoded by Tomato leaf curl Yunnan virus interferes with mitogen-activated protein kinase cascade-related defense responses through inhibiting the dissociation of the ERECTA/BKI1 complex.

Mitogen-activated protein kinase (MAPK) cascades are involved in host defense against pathogens and are often activated by upstream plasma membrane leucine-rich repeat receptor-like kinases (LRR-RLKs). ERECTA (ER) is an LRR-RLK that regulates plant developmental processes through activating MAPK cascades. Tomato leaf curl Yunnan virus (TLCYnV) C4 protein interacts with BKI1, stabilizes it at the plasma membrane and impairs ER autophosphorylation through suppressing the dissociation of the BKI1/ER complex, and then inhibits the activation of downstream MAPK cascades, which ultimately creates a favorable environment for TLCYnV infection. This study provides a novel viral strategy to impair MAPK activation.

Cytoplasmic and nuclear Sw-5b NLR act both independently and synergistically to confer full host defense against tospovirus infection.

Plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors play critical roles in mediating host immunity to pathogen attack. We use tomato Sw-5b::tospovirus as a model system to study the specific role of the compartmentalized plant NLR in dictating host defenses against the virus at different infection steps. We demonstrated here that tomato NLR Sw-5b distributes to the cytoplasm and nucleus, respectively, to play different roles in inducing host resistances against tomato spotted wilt orthotospovirus (TSWV) infection. The cytoplasmic-enriched Sw-5b induces a strong cell death response to inhibit TSWV replication. This host response is, however, insufficient to block viral intercellular and long-distance movement. The nuclear-enriched Sw-5b triggers a host defense that weakly inhibits viral replication but strongly impedes virus intercellular and systemic movement. Furthermore, the cytoplasmic and nuclear Sw-5b act synergistically to dictate a full host defense of TSWV infection. We further demonstrated that the extended N-terminal Solanaceae domain (SD) of Sw-5b plays critical roles in cytoplasm/nucleus partitioning. Sw-5b NLR controls its cytoplasm localization. Strikingly, the SD but not coil-coil domain is crucial for Sw-5b receptor to import into the nucleus to trigger the immunity. The SD was found to interact with importins. Silencing both importin α and ß expression disrupted Sw-5b nucleus import and host immunity against TSWV systemic infection. Collectively, our findings suggest that Sw-5b bifurcates disease resistances by cytoplasm/nucleus partitioning to block different infection steps of TSWV. The findings also identified a new regulatory role of extra domain of a plant NLR in mediating host innate immunity.

Tomato leaf curl Yunnan virus-encoded C4 induces cell division through enhancing stability of Cyclin D 1.1 via impairing NbSKη -mediated phosphorylation in Nicotiana benthamiana.

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.

Geminivirus C4 antagonizes the HIR1-mediated hypersensitive response by inhibiting the HIR1 self-interaction and promoting degradation of the protein.

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.

Identification of an RNA silencing suppressor encoded by a mastrevirus.

Wheat dwarf virus (WDV) is a DNA virus belonging to the genus Mastrevirus of the family Geminiviridae. In this study, we report that the Rep protein encoded by WDV is a RNA silencing supressor as determined by co-infiltration assays using transgenic Nicotiana benthamiana line 16c carrying the GFP reporter gene. The Rep protein was shown to inhibit both local and systemic RNA silencing of the GFP gene as well as the spread of systemic GFP RNA silencing signals. Gel mobility shift assays showed that the Rep protein binds 21 nt and 24 nt small interfering RNA (siRNA) duplexes and single-stranded (ss)-siRNA. To our knowledge, this is the first identification of an RNA silencing suppressor encoded by mastreviruses. Furthermore, deletion mutagenesis indicates that both the N- and C-terminal regions of the Rep protein are not critical for silencing suppression and self-interaction, but the N terminus of Rep is necessary for its pathogenicity.

Two viral proteins translated from one open reading frame target different layers of plant defense.

Multilayered defense responses are activated upon pathogen attack. Viruses utilize a number of strategies to maximize the coding capacity of their small genomes and produce viral proteins for infection, including suppression of host defense. Here, we reveal translation leakage as one of these strategies: two viral effectors encoded by tomato golden mosaic virus, chloroplast-localized C4 (cC4) and membrane-associated C4 (mC4), are translated from two in-frame start codons and function cooperatively to suppress defense. cC4 localizes in chloroplasts, to which it recruits NbPUB4 to induce ubiquitination of the outer membrane; as a result, this organelle is degraded, and chloroplast-mediated defenses are abrogated. However, chloroplast-localized cC4 induces the production of singlet oxygen (1O2), which in turn promotes translocation of the 1O2 sensor NbMBS1 from the cytosol to the nucleus, where it activates expression of the CERK1 gene. Importantly, an antiviral effect exerted by CERK1 is countered by mC4, localized at the plasma membrane. mC4, like cC4, recruits NbPUB4 and promotes the ubiquitination and subsequent degradation of CERK1, suppressing membrane-based, receptor-like kinase-dependent defenses. Importantly, this translation leakage strategy seems to be conserved in multiple viral species and is related to host range. This finding suggests that stacking of different cellular antiviral responses could be an effective way to abrogate viral infection and engineer sustainable resistance to major crop viral diseases in the field.

Molecular characterization and pathogenicity of an infectious clone of tomato leaf curl New Delhi virus isolate infecting Cucumis melo.

Tomato leaf curl New Delhi virus (ToLCNDV) is a member of the genus Begomovirus, and causes devastating disease in the world. In recent years, ToLCNDV was rapidly spreading in China and induces severe economic losses in agriculture. In this study, we sequenced and characterized the complete genome of ToLCNDV isolates from melon plants showing leaf curling and stunting symptoms in Jiangsu Province of China. We constructed a full-length infectious cDNA clone of ToLCNDV, which could induce systemic infection with typical symptoms in Nicotiana benthamiana, Citrullus melo, and Citrullus lanatus plants through agrobacterium-mediated inoculation. Further experimental evidence demonstrated that the virions produced in plants infected with the infectious clone of ToLCNDV are biologically active and sap-transmissible. We also evaluated the resistance of commercial melon cultivars to ToLCNDV and found all testing melon cultivars were susceptible to ToLCNDV. Collectively, the reverse genetic system developed herein will facilitate further research on biological functions of proteins encoded by ToLCNDV and plant-ToLCNDV interactions, which might provide new insights into breeding resistance germplasm in crops.

Tomato leaf curl New Delhi virus: an emerging plant begomovirus threatening cucurbit production.

Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, was first reported to infect tomato and has recently spread rapidly as an emerging disease to Cucurbitaceae crops. To date, the virus has been reported to infect more than 11 cucurbit crops, in 16 countries and regions, causing severe yield losses. In autumn 2022, ToLCNDV was first isolated from cucurbit plants in Southeastern coastal areas of China. Phylogenetic analysis established that these isolates belong to the Asian ToLCNDV clade, and shared high nucleotide identity and closest genetic relationship with the DNA-A sequence from the Chinese tomato-infecting ToLCNDV isolate (Accession no. OP356207) and the tomato New Delhi ToLCNDV-Severe isolate (Accession no. HM159454). In this review, we summarize the occurrence and distribution, host range, detection and diagnosis, control strategies, and genetic resistance of ToLCNDV in the Cucurbitaceae. We then summarize pathways that could be undertaken to improve our understanding of this emerging disease, with the objective to develop ToLCNDV-resistant cucurbit cultivars. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-023-00118-4.

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

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 NbSKη, 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 NbSKη 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 NbSKη. TLCYnV C4 is phosphorylated by NbSKη in the nucleus, which promotes myristoylation of the viral protein. Myristoylation of phosphorylated C4 favors its interaction with exportin-α (XPO I), which in turn facilitates nuclear export of the C4/NbSKη complex. Supporting this model, chemical inhibition of N-myristoyltransferases or exportin-α 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 NbSKη, myristoylation, and interaction with exportin-α, is critical for its function as a pathogenicity factor.