Molecular basis of methyl-salicylate-mediated plant airborne defence.

Aphids transmit viruses and are destructive crop pests1. Plants that have been attacked by aphids release volatile compounds to elicit airborne defence (AD) in neighbouring plants2-5. However, the mechanism underlying AD is unclear. Here we reveal that methyl-salicylate (MeSA), salicylic acid-binding protein-2 (SABP2), the transcription factor NAC2 and salicylic acid-carboxylmethyltransferase-1 (SAMT1) form a signalling circuit to mediate AD against aphids and viruses. Airborne MeSA is perceived and converted into salicylic acid by SABP2 in neighbouring plants. Salicylic acid then causes a signal transduction cascade to activate the NAC2-SAMT1 module for MeSA biosynthesis to induce plant anti-aphid immunity and reduce virus transmission. To counteract this, some aphid-transmitted viruses encode helicase-containing proteins to suppress AD by interacting with NAC2 to subcellularly relocalize and destabilize NAC2. As a consequence, plants become less repellent to aphids, and more suitable for aphid survival, infestation and viral transmission. Our findings uncover the mechanistic basis of AD and an aphid-virus co-evolutionary mutualism, demonstrating AD as a potential bioinspired strategy to control aphids and viruses.

A viral protein disrupts vacuolar acidification to facilitate virus infection in plants.

Vacuolar acidification is essential for vacuoles in diverse physiological functions. However, its role in plant defense, and whether and how pathogens affect vacuolar acidification to promote infection remain unknown. Here, we show that Barley stripe mosaic virus (BSMV) replicase γa, but not its mutant γaR569A , directly blocks acidification of vacuolar lumen and suppresses autophagic degradation to promote viral infection in plants. These were achieved via molecular interaction between γa and V-ATPase catalytic subunit B2 (VHA-B2), leading to disruption of the interaction between VHA-B2 and V-ATPase catalytic subunit E (VHA-E), which impairs the membrane localization of VHA-B2 and suppresses V-ATPase activity. Furthermore, a mutant virus BSMVR569A with the R569A point mutation possesses less viral pathogenicity. Interestingly, multiple viral infections block vacuolar acidification. These findings reveal that functional vacuolar acidification is required for plant antiviral defense and disruption of vacuolar acidification could be a general viral counter-defense strategy employed by multiple viruses.

Coupled Stable Isotope Tracing and Sulfamic Acid Reduction (SIT-SAR) Method to Determine the Ammonia and Nitrite Oxidation Rates in Water and Sediments.

Herein, a method was developed to measure the ammonia oxidation rate (Ra) and the nitrite oxidation rate (Rn) of water and sediment samples using a coupled stable isotope tracing and sulfamic acid reduction (SIT-SAR) method. 15NH4+ was used as a tracer to determine the ammonia oxidation rates (Ra) by calculating the concentrations of produced 15NO2- and 15NO3- during incubation, while 15NO2- was used as a tracer to determine the nitrite oxidation rates (Rn) by calculating the increase of 15NO3- during incubation. 15NO2- was chemically reduced to 29N2 with 15 mmol·L-1 sulfamic acid (SA). 15NO3- was first reduced to 15NO2- with a zinc-cadmium reducing agent, and then 15NO2- was subsequently reduced to 29N2 with SA. The produced 29N2 was measured by a membrane inlet mass spectrometer (MIMS). Under optimized experimental conditions, this method provides a sensitive (detection limit: 0.5 µmol·L-1) and precise (relative standard deviation: 4.80% for 15NO2-, 3.82% for 15NO3-) approach to quantify the concentrations of 15NO2- (0.5-150 µmol·L-1) and 15NO3- (0.5-120 µmol·L-1) in water and sediment samples over a wide range of salinities (0-30‰) with excellent calibration curves (R2 ≥ 0.999). This method was a successful application to estuarine water and sediments along the salinity gradient. Overall, the SIT-SAR method provided a rapid, accurate, and cost-effective means to determine Ra and Rn simultaneously.

Ethylene-MPK8-ERF.C1-PR module confers resistance against Botrytis cinerea in tomato fruit without compromising ripening.

The plant hormone ethylene plays a critical role in fruit defense against Botrytis cinerea attack, but the underlying mechanisms remain poorly understood. Here, we showed that ethylene response factor SlERF.C1 acts as a key regulator to trigger the ethylene-mediated defense against B. cinerea in tomato fruits without compromising ripening. Knockout of SlERF.C1 increased fruit susceptibility to B. cinerea with no effect on ripening process, while overexpression enhanced resistance. RNA-Seq, transactivation assays, EMSA and ChIP-qPCR results indicated that SlERF.C1 activated the transcription of PR genes by binding to their promoters. Moreover, SlERF.C1 interacted with the mitogen-activated protein kinase SlMPK8 which allowed SlMPK8 to phosphorylate SlERF.C1 at the Ser174 residue and increases its transcriptional activity. Knocking out of SlMPK8 increased fruit susceptibility to B. cinerea, whereas overexpression enhanced resistance without affecting ripening. Furthermore, genetic crosses between SlMPK8-KO and SlERF.C1-OE lines reduced the resistance to B. cinerea attack in SlERF.C1-OE fruits. In addition, B. cinerea infection induced ethylene production which in turn triggered SlMPK8 transcription and enhanced the phosphorylation of SlERF.C1. Overall, our findings reveal the regulatory mechanism of the 'Ethylene-MPK8-ERF.C1-PR' module in resistance against B. cinerea and provide new insight into the manipulation of gray mold disease in fruits.

ß-1,3-GLUCANASE10 regulates tomato development and disease resistance by modulating callose deposition.

ß-1,3-Glucanases are considered key regulators responsible for the degradation of callose in plants, yet little is known about the role and mode of action of their encoding genes in tomato (Solanum lycopersicum). In the present study, we identified the ß-1,3-glucanase encoding gene ß-1,3-GLUCANASE10 (SlBG10) and revealed its regulation in tomato pollen and fruit development, seed production, and disease resistance by modulating callose deposition. Compared with wild-type (WT) or SlBG10 overexpressing (SlBG10-OE) lines, knockout of SlBG10 caused pollen arrest and failure to set fruit with reduced male rather than female fecundity. Further analyses showed that SlBG10-knockout promoted callose deposition in anther at the tetrad-to-microspore stages, resulting in pollen abortion and male sterility. Moreover, loss-of-function SlBG10 delayed degradation of endosperm cell wall calloses during cellularization and impeded early seed development. We also uncovered that Botrytis cinerea infection induces SlBG10 expression in WT tomato, and the knockout lines showed increased callose accumulation in fruit pericarps, reduced susceptibility to B. cinerea, and enhanced antioxidant capacity to maintain tomato fruit quality. However, the expression of genes encoding cell wall hydrolases decreased in SlBG10-knockout tomatoes and thus led to an increase in pericarp epidermal thickness, enhancement in fruit firmness, reduction of fruit water loss, and extension of tomato shelf life. These findings not only expand our understanding of the involvement of ß-1,3-glucanases as callose regulators in multiple developmental processes and pathogen resistance but also provide additional insight into the manipulation of multiagronomic traits for targeted tomato breeding.

Cotton leaf curl Multan virus C4 protein suppresses autophagy to facilitate viral infection.

Autophagy plays an important role in plant antiviral defense. Several plant viruses are reported to encode viral suppressor of autophagy (VSA) to prevent autophagy for effective virus infection. However, whether and how other viruses, in particular DNA viruses, also encode VSAs to affect viral infection in plants is unknown. Here, we report that the C4 protein encoded by Cotton leaf curl Multan geminivirus (CLCuMuV) inhibits autophagy by binding to the autophagy negative regulator eukaryotic translation initiation factor 4A (eIF4A) to enhance the eIF4A-Autophagy-related protein 5 (ATG5) interaction. By contrast, the R54A or R54K mutation in C4 abolishes its capacity to interact with eIF4A, and neither C4R54A nor C4R54K can suppress autophagy. However, the R54 residue is not essential for C4 to interfere with transcriptional gene silencing or post-transcriptional gene silencing. Moreover, plants infected with mutated CLCuMuV-C4R54K develop less severe symptoms with decreased levels of viral DNA. These findings reveal a molecular mechanism underlying how the DNA virus CLCuMuV deploys a VSA to subdue host cellular antiviral autophagy defense and uphold viral infection in plants.

A comprehensive metabolic map reveals major quality regulations in red-flesh kiwifruit (Actinidia chinensis).

Kiwifruit (Actinidia chinensis) is one of the popular fruits world-wide, and its quality is mainly determined by key metabolites (sugars, flavonoids, and vitamins). Previous works on kiwifruit are mostly done via a single omics approach or involve only limited metabolites. Consequently, the dynamic metabolomes during kiwifruit development and ripening and the underlying regulatory mechanisms are poorly understood. In this study, using high-resolution metabolomic and transcriptomic analyses, we investigated kiwifruit metabolic landscapes at 11 different developmental and ripening stages and revealed a parallel classification of 515 metabolites and their co-expressed genes into 10 distinct metabolic vs gene modules (MM vs GM). Through integrative bioinformatics coupled with functional genomic assays, we constructed a global map and uncovered essential transcriptomic and transcriptional regulatory networks for all major metabolic changes that occurred throughout the kiwifruit growth cycle. Apart from known MM vs GM for metabolites such as soluble sugars, we identified novel transcription factors that regulate the accumulation of procyanidins, vitamin C, and other important metabolites. Our findings thus shed light on the kiwifruit metabolic regulatory network and provide a valuable resource for the designed improvement of kiwifruit quality.

Cotton leaf curl Multan virus ßC1 Protein Induces Autophagy by Disrupting the Interaction of Autophagy-Related Protein 3 with Glyceraldehyde-3-Phosphate Dehydrogenases.

Autophagy plays an important role in plant-pathogen interactions. Several pathogens including viruses induce autophagy in plants, but the underpinning mechanism remains largely unclear. Furthermore, in virus-plant interactions, viral factor(s) that induce autophagy have yet to be identified. Here, we report that the ßC1 protein of Cotton leaf curl Multan betasatellite (CLCuMuB) interacts with cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC), a negative autophagic regulator, to induce autophagy in Nicotiana benthamiana CLCuMuB ßC1 bound to GAPCs and disrupted the interaction between GAPCs and autophagy-related protein 3 (ATG3). A mutant ßC1 protein (ßC13A) in which I45, Y48, and I53 were all substituted with Ala (A), had a dramatically reduced binding capacity with GAPCs, failed to disrupt the GAPCs-ATG3 interactions and failed to induce autophagy. Furthermore, mutant virus carrying ßC13A showed increased symptoms and viral DNA accumulation associated with decreased autophagy in plants. These results suggest that CLCuMuB ßC1 activates autophagy by disrupting GAPCs-ATG3 interactions.

Discrepant Effects of Flooding on Assembly Processes of Abundant and Rare Communities in Riparian Soils.

Numerous rare species coexist with a few abundant species in microbial communities and together play an essential role in riparian ecosystems. Relatively little is understood, however, about the nature of assembly processes of these communities and how they respond to a fluctuating environment. In this study, drivers controlling the assembly of abundant and rare subcommunities for bacteria and archaea in a riparian zone were determined, and their resulting patterns on these processes were analyzed. Abundant and rare bacteria and archaea showed a consistent variation in the community structure along the riparian elevation gradient, which was closely associated with flooding frequency. The community assembly of abundant bacteria was not affected by any measured environmental variables, while soil moisture and ratio of submerged time to exposed time were the two most decisive factors determining rare bacterial community. Assembly of abundant archaeal community was also determined by these two factors, whereas rare archaea was significantly associated with soil carbon-nitrogen ratio and total carbon content. The assembly process of abundant and rare bacterial subcommunities was driven respectively by dispersal limitation and variable selection. Undominated processes and dispersal limitation dominated the assembly of abundant archaea, whereas homogeneous selection primarily driven rare archaea. Flooding may therefore play a crucial role in determining the community assembly processes by imposing disturbances and shaping soil niches. Overall, this study reveals the assembly patterns of abundant and rare communities in the riparian zone and provides further insight into the importance of their respective roles in maintaining a stable ecosystem during times of environmental perturbations.

Spinach-based RNA mimicking GFP in plant cells.

Spinach RNA-mimicking GFP (S-RMG) has been successfully used to monitor cellular RNAs including microRNAs in bacterium, yeast, and human cells. However, S-RMG has not been established in plants. In this study, we found that like bacterial, yeast, and human cellular tRNAs, plant tRNAs such as tRNALys can protect and/or stabilize the Spinach RNA aptamer interaction with the fluorophore DFHBI enabling detectable levels of green fluorescence to be emitted. The tRNALys-Spinach-tRNALys, once delivered into "chloroplast-free" onion epidermal cells can emit strong green fluorescence in the presence of DFHBI. Our results demonstrate for the first time that Spinach-based RNA visualization has the potential for in vivo monitoring of RNAs in plant cells.

Plant NLR immune receptor Tm-22 activation requires NB-ARC domain-mediated self-association of CC domain.

The nucleotide-binding, leucine-rich repeat-containing (NLR) class of immune receptors of plants and animals recognize pathogen-encoded proteins and trigger host defenses. Although animal NLRs form oligomers upon pathogen recognition to activate downstream signaling, the mechanisms of plant NLR activation remain largely elusive. Tm-22 is a plasma membrane (PM)-localized coiled coil (CC)-type NLR and confers resistance to Tobacco mosaic virus (TMV) by recognizing its viral movement protein (MP). In this study, we found that Tm-22 self-associates upon recognition of MP. The CC domain of Tm-22 is the signaling domain and its function requires PM localization and self-association. The nucleotide-binding (NB-ARC) domain is important for Tm-22 self-interaction and regulates activation of the CC domain through its nucleotide-binding and self-association. (d)ATP binding may alter the NB-ARC conformation to release its suppression of Tm-22 CC domain-mediated cell death. Our findings provide the first example of signaling domain for PM-localized NLR and insight into PM-localized NLR activation.

Plant UVRAG interacts with ATG14 to regulate autophagosome maturation and geminivirus infection.

Autophagy is an essential degradation pathway that assists eukaryote survival under multiple stress conditions. Autophagosomes engulfing cargoes accomplish degradation only when they have matured through fusing with lysosomes or vacuoles. However, the molecular machinery mediating autophagosome maturation in plants remains unknown. Using the combined approaches of mass spectrometry, biochemistry, reverse genetics and microscopy, we uncover that UVRAG, a subunit of the class III phosphatidylinositol 3-kinase complexes in Nicotiana benthamiana, plays an essential role in autophagsome maturation via ATG14-assisted recruitment to autophagosomes and by facilitating RAB7 activation. An interaction between N. benthamiana UVRAG and ATG14 was observed in vitro and in vivo, which strikingly differed from their mutually exclusive appearance in different PI3KC3 complexes in yeast and mammals. This interaction increased the localisation of UVRAG on autophagosomes and enabled the convergence of autophagic and late endosomal structures, where they contributed to fusions between these two types of organelles by recruiting the essential membrane fusion factors RAB7 GTPase and the homotypic fusion and protein sorting (HOPS) complex. In addition, we uncovered a joint contribution of ATG14 and UVRAG to geminiviral infection, beyond autophagy. Our study provides insights into the mechanisms of autophagosome maturation in plants and expands the understanding of organisations and roles of the PI3KC3 complexes.

Efficient and high-throughput pseudorecombinant-chimeric Cucumber mosaic virus-based VIGS in maize.

Virus-induced gene silencing (VIGS) is a versatile and attractive approach for functional gene characterization in plants. Although several VIGS vectors for maize (Zea mays) have been previously developed, their utilities are limited due to low viral infection efficiency, insert instability, short maintenance of silencing, inadequate inoculation method, or abnormal requirement of growth temperature. Here, we established a Cucumber mosaic virus (CMV)-based VIGS system for efficient maize gene silencing that overcomes many limitations of VIGS currently available for maize. Using two distinct strains, CMV-ZMBJ and CMV-Fny, we generated a pseudorecombinant-chimeric (Pr) CMV. Pr CMV showed high infection efficacy but mild viral symptoms in maize. We then constructed Pr CMV-based vectors for VIGS, dubbed Pr CMV VIGS. Pr CMV VIGS is simply performed by mechanical inoculation of young maize leaves with saps of Pr CMV-infected Nicotiana benthamiana under normal growth conditions. Indeed, suppression of isopentenyl/dimethylallyl diphosphate synthase (ZmIspH) expression by Pr CMV VIGS resulted in non-inoculated leaf bleaching as early as 5 d post-inoculation (dpi) and exhibited constant and efficient systemic silencing over the whole maize growth period up to 105 dpi. Furthermore, utilizing a ligation-independent cloning (LIC) strategy, we developed a modified Pr CMV-LIC VIGS vector, allowing easy gene cloning for high-throughput silencing in maize. Thus, our Pr CMV VIGS system provides a much-improved toolbox to facilitate efficient and long-duration gene silencing for large-scale functional genomics in maize, and our pseudorecombination-chimera combination strategy provides an approach to construct efficient VIGS systems in plants.

Diverse nirS-type Denitrifying Bacteria Contribute to Vital Nitrogen Loss in Natural Acidic Red Soils.

Denitrifying bacteria, playing a key role in nitrogen removal in ecosystem, are highly diverse and complex in their community composition. However, there were few reports on the abundance, community composition, and the contribution to nitrogen loss of denitrifiers in natural acidic red soils. In this study, we investigated the structure and function of nirS-type denitrifying bacteria in ten natural red soil samples collected from nine provinces in southern China, based on quantitative polymerase chain reaction (qPCR) and high-throughput sequencing techniques. Nitrogen loss from microbial denitrification in red soils of southern China was estimated up to 9.86 Tg N per year based on 15N isotope tracing method. The abundance of nirS-type denitrifiers varied from 8.41 × 105 to 2.55 × 109 copies per gram of dry weight. The community of nirS-type denitrifying bacterial was revealed, which contained 50 dominant OTUs assigned to 9 clusters phylogenetically related to Marinobacter, Rhodobacter, and other uncultured species. pH was the key factor affecting both denitrification rates and community composition. Our results demonstrate that nirS-type denitrifying bacteria have higher abundance, diversity, and contribution to the nitrogen loss in natural acidic red soils of southern China.

The miR157-SPL-CNR module acts upstream of bHLH101 to negatively regulate iron deficiency responses in tomato.

Iron (Fe) homeostasis is critical for plant growth, development, and stress responses. Fe levels are tightly controlled by intricate regulatory networks in which transcription factors (TFs) play a central role. A series of basic helix-loop-helix (bHLH) TFs have been shown to contribute to Fe homeostasis, but the regulatory layers beyond bHLH TFs remain largely unclear. Here, we demonstrate that the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) TF SlSPL-CNR negatively regulates Fe-deficiency responses in tomato (Solanum lycopersicum) roots. Fe deficiency rapidly repressed the expression of SlSPL-CNR, and Fe deficiency responses were intensified in two clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-generated SlSPL-CNR knock-out lines compared to the wild-type. Comparative transcriptome analysis identified 47 Fe deficiency-responsive genes the expression of which is negatively regulated by SlSPL-CNR, one of which, SlbHLH101, helps regulate Fe uptake genes. SlSPL-CNR localizes the nucleus and interacts with the GTAC and BOX 4 (ATTAAT) motifs in the SlbHLH101 promoter to repress its expression. Inhibition of SlSPL-CNR expression in response to Fe deficiency was well correlated with the expression of the microRNA SlymiR157. SlymiR157-overexpressing tomato lines displayed enhanced Fe deficiency responses, as did SlSPL-CNR loss-of-function mutants. We propose that the SlymiR157-SlSPL-CNR module represents a novel pathway that acts upstream of SlbHLH101 to regulate Fe homeostasis in tomato roots.

METHYLTRANSFERASE1 and Ripening Modulate Vivipary during Tomato Fruit Development.

Vivipary, wherein seeds germinate prior to dispersal while still associated with the maternal plant, is an adaptation to extreme environments. It is normally inhibited by the establishment of dormancy. The genetic framework of vivipary has been well studied; however, the role of epigenetics in vivipary remains unknown. Here, we report that silencing of METHYLTRANSFERASE1 (SlMET1) promoted precocious seed germination and seedling growth within the tomato (Solanum lycopersicum) epimutant Colorless non-ripening (Cnr) fruits. This was associated with decreases in abscisic acid concentration and levels of mRNA encoding 9-cis-epoxycarotenoid-dioxygenase (SlNCED), which is involved in abscisic acid biosynthesis. Differentially methylated regions were identified in promoters of differentially expressed genes, including SlNCED SlNCED knockdown also induced viviparous seedling growth in Cnr fruits. Strikingly, Cnr ripening reversion suppressed vivipary. Moreover, neither SlMET1/SlNCED-virus-induced gene silencing nor transgenic SlMET1-RNA interference produced vivipary in wild-type tomatoes; the latter affected leaf architecture, arrested flowering, and repressed seed development. Thus, a dual pathway in ripening and SlMET1-mediated epigenetics coordinates the blockage of seed vivipary.

Spatial variability pattern of the anaerobic ammonia-oxidizing bacterial community across a salinity gradient from river to ocean.

In natural habitats, the diversity of anaerobic ammonia-oxidizing (anammox) bacteria could be affected by multiple environmental variables. In this study, we investigated the distribution of the anammox bacterial community in surface sediment from the Dongjiang River (riverine sediment, DJ) to the Pearl River Estuary (estuarine sediment, PRE) and then to the South China Sea (coastal sediment, SCS). The results revealed evident differences in the structural diversity of anammox bacteria in three different habitats. Candidatus Brocadia accounted for approximately 90% of the total anammox bacteria in DJ, conversely, Ca. Scalindua dominated in the SCS. Nevertheless, Ca. Scalindua, Ca. Brocadia and Ca. Kuenenia coexisted in the PRE. The qPCR results indicated that anammox bacterial 16S rRNA gene abundance ranged from 2.23 × 105 to 1.19 × 107 copies g-1 of wet weight, but no significant correlation was found between the abundances and environmental variables (p > 0.05). The relative abundances of Ca. Brocadia gradually decreased with increasing salinity, and Ca. Scalindua showed the opposite trend, suggesting that salinity was a crucial factor in sculpturing the community composition of anammox bacteria in natural environments. Ca. Brocadia should be able to live in freshwater ecosystems, but it can also tolerate a certain level of salinity. Ca. Scalindua was halophilic anammox bacterium and exists only in saline environments. Ca. Kuenenia could adapt to a wide range of salinity and preferred to live in high DIN level conditions according to our search. The distribution pattern of anammox bacteria may be the result of microbial migration and long-term adaptation to salinity.

Shift of DNRA bacterial community composition in sediment cores of the Pearl River Estuary and the impact of environmental factors.

Dissimilatory nitrate reduction to ammonia (DNRA) process, competing with denitrification and anaerobic ammonia oxidation (anammox) for nitrate, is an important nitrogen retention pathway in the environment. Previous studies on DNRA bacterial diversity and composition focused on the surface sediments in estuaries, but studies on the deep sediments are limited, and the linkage between DNRA community structure and complex estuarine environment remains unclear. In this study, through high-throughput sequencing of nrfA gene followed by high-resolution sample inference, we examined spatially and temporally the composition and diversity of DNRA bacteria along a salinity gradient in five sediment cores of the Pearl River Estuary (PRE). We found a higher diversity and richness of DNRA bacteria in sediments with lower organic carbon, where sea water intersects fresh water. Moreover, the DNRA bacterial communities had the specific spatially distribution coupling with their metabolic difference along the salinity gradient of the Pearl River Estuary, but no obvious difference along the sediment depth. The distribution of DNRA bacteria in the PRE was largely driven by various environmental factors, including salinity, Oxidation-Reduction Potential (ORP), ammonium, nitrate and Corg/NO3-. Furthermore, dominant DNRA bacteria were found to be the key populations of DNRA communities in the PRE sediments by network analysis. Collectively, our results showed that niche difference of DNRA bacteria indeed occurs in the Pearl River Estuary.

An RNAi suppressor activates in planta virus-mediated gene editing.

RNA-guided CRISPR/Cas9 technology has been developed for gene/genome editing (GE) in organisms across kingdoms. However, in planta delivery of the two core GE components, Cas9 and small guide RNA (sgRNA), often involves time-consuming and labor-intensive production of transgenic plants. Here we show that Foxtail mosaic virus, a monocot- and dicot-infecting potexvirus, can simultaneously express Cas9, sgRNA, and an RNAi suppressor to efficiently induce GE in Nicotiana benthamiana through a transgenic plant-free manner.

Geminiviral V2 Protein Suppresses Transcriptional Gene Silencing through Interaction with AGO4.

In plants, RNA-directed DNA methylation (RdDM)-mediated transcriptional gene silencing (TGS) is a natural antiviral defense against geminiviruses. Several geminiviral proteins have been shown to target the enzymes related to the methyl cycle or histone modification; however, it remains largely unknown whether and by which mechanism geminiviruses directly inhibit RdDM-mediated TGS. In this study, we showed that Cotton leaf curl Multan virus (CLCuMuV) V2 directly interacts with Nicotiana benthamiana AGO4 (NbAGO4) and that the L76S mutation in V2 (V2L76S) abolishes such interaction. We further showed that V2, but not V2L76S, can suppresses RdDM and TGS. Silencing of NbAGO4 inhibits TGS, reduces the viral methylation level, and enhances CLCuMuV DNA accumulation. In contrast, the V2L76S substitution mutant attenuates CLCuMuV infection and enhances the viral methylation level. These findings reveal that CLCuMuV V2 contributes to viral infection by interaction with NbAGO4 to suppress RdDM-mediated TGS in plants.IMPORTANCE In plants, the RNA-directed DNA methylation (RdDM) pathway is a natural antiviral defense mechanism against geminiviruses. However, how geminiviruses counter RdDM-mediated defense is largely unknown. Our findings reveal that Cotton leaf curl Multan virus V2 contributes to viral infection by interaction with NbAGO4 to suppress RNA-directed DNA methylation-mediated transcriptional gene silencing in plants. Our work provides the first evidence that a geminiviral protein is able to directly target core RdDM components to counter RdDM-mediated TGS antiviral defense in plants, which extends our current understanding of viral counters to host antiviral defense.