Antiviral ARGONAUTEs Against Turnip Crinkle Virus Revealed by Image-Based Trait Analysis.

RNA-based silencing functions as an important antiviral immunity mechanism in plants. Plant viruses evolved to encode viral suppressors of RNA silencing (VSRs) that interfere with the function of key components in the silencing pathway. As effectors in the RNA silencing pathway, ARGONAUTE (AGO) proteins are targeted by some VSRs, such as that encoded by Turnip crinkle virus (TCV). A VSR-deficient TCV mutant was used to identify AGO proteins with antiviral activities during infection. A quantitative phenotyping protocol using an image-based color trait analysis pipeline on the PlantCV platform, with temporal red, green, and blue imaging and a computational segmentation algorithm, was used to measure plant disease after TCV inoculation. This process captured and analyzed growth and leaf color of Arabidopsis (Arabidopsis thaliana) plants in response to virus infection over time. By combining this quantitative phenotypic data with molecular assays to detect local and systemic virus accumulation, AGO2, AGO3, and AGO7 were shown to play antiviral roles during TCV infection. In leaves, AGO2 and AGO7 functioned as prominent nonadditive, anti-TCV effectors, whereas AGO3 played a minor role. Other AGOs were required to protect inflorescence tissues against TCV. Overall, these results indicate that distinct AGO proteins have specialized, modular roles in antiviral defense across different tissues, and demonstrate the effectiveness of image-based phenotyping to quantify disease progression.

COP1, a negative regulator of photomorphogenesis, positively regulates plant disease resistance via double-stranded RNA binding proteins.

The E3 ubiquitin ligase COP1 (Constitutive Photomorphogenesis 1) is a well known component of the light-mediated plant development that acts as a repressor of photomorphogenesis. Here we show that COP1 positively regulates defense against turnip crinkle virus (TCV) and avrRPM1 bacteria by contributing to stability of resistance (R) protein HRT and RPM1, respectively. HRT and RPM1 levels and thereby pathogen resistance is significantly reduced in the cop1 mutant background. Notably, the levels of at least two double-stranded RNA binding (DRB) proteins DRB1 and DRB4 are reduced in the cop1 mutant background suggesting that COP1 affects HRT stability via its effect on the DRB proteins. Indeed, a mutation in either drb1 or drb4 resulted in degradation of HRT. In contrast to COP1, a multi-subunit E3 ligase encoded by anaphase-promoting complex (APC) 10 negatively regulates DRB4 and TCV resistance but had no effect on DRB1 levels. We propose that COP1-mediated positive regulation of HRT is dependent on a balance between COP1 and negative regulators that target DRB1 and DRB4.

A nuclear fraction of turnip crinkle virus capsid protein is important for elicitation of the host resistance response.

The N-terminal 25 amino acids (AAs) of turnip crinkle virus (TCV) capsid protein (CP) are recognized by the resistance protein HRT to trigger a hypersensitive response (HR) and systemic resistance to TCV infection. This same region of TCV CP also contains a motif that interacts with the transcription factor TIP, as well as a nuclear localization signal (NLS). However, it is not yet known whether nuclear localization of TCV CP is needed for the induction of HRT-mediated HR and resistance. Here we present new evidence suggesting a tight correlation between nuclear inclusions formed by CP and the manifestation of HR. We show that a fraction of TCV CP localized to cell nuclei to form discrete inclusion-like structures, and a mutated CP (R6A) known to abolish HR failed to form nuclear inclusions. Notably, TIP-CP interaction augments the inclusion-forming activity of CP by tethering inclusions to the nuclear membrane. This TIP-mediated augmentation is also critical for HR resistance, as another CP mutant (R8A) known to elicit a less restrictive HR, though still self-associated into nuclear inclusions, failed to direct inclusions to the nuclear membrane due to its inability to interact with TIP. Finally, exclusion of CP from cell nuclei abolished induction of HR. Together, these results uncovered a strong correlation between nuclear localization and nuclear inclusion formation by TCV CP and induction of HR, and suggest that CP nuclear inclusions could be the key trigger of the HRT-dependent, yet TIP-reinforced, resistance to TCV.

A spectrum of HRT-dependent hypersensitive responses elicited by the 52 amino acid N-terminus of turnip crinkle virus capsid protein and its mutants.

The capsid protein (CP) of turnip crinkle virus (TCV) is the elicitor of hypersensitive response (HR) and resistance mediated by the resistance protein HRT in the Di-17 ecotype of Arabidopsis. Here we identified the N-terminal 52-amino-acid R domain of TCV CP as the elicitor of HRT-dependent HR in Nicotiana benthamiana. Mutating this domain at position 6 (R6A), but not at positions 8 (R8A) or 14 (G14A), abolished HR in N. benthamiana. However, on Di-17 Arabidopsis leaves only R8A R domain elicited visible epidermal HR. When incorporated in infectious TCV RNAs, R8A and G14A mutations exerted dramatically different effects in Di-17 plants, as R8A caused systemic cell death whereas G14A led to complete restriction of the mutant virus. This continual spectrum of HR and resistance responses elicited by various R domain mutants suggests that the CP-HRT interaction could be perturbed by conformational changes in the R domain of TCV CP.

RNA silencing components mediate resistance signaling against turnip crinkle virus.

Species-specific immunity is induced when an effector protein from a specific pathogen strain is perceived by a cognate resistance (R) protein in the plant. In Arabidopsis, the R protein HRT, which confers resistance to turnip crinkle virus (TCV), is activated upon recognition of the TCV coat-protein (CP), a potent suppressor of host RNA silencing. Recognition by HRT does not require RNA silencing suppressor function of CP and is not associated with the accumulation of TCV-specific small-RNA. However, several components of the host RNA silencing pathway participate in HRT-mediated defense against TCV. For example, the double stranded RNA binding protein (DRB) 4 interacts with the plasma membrane localized HRT, and is required for its stability. Intriguingly, TCV infection promotes the cytosolic accumulation of the otherwise primarily nuclear DRB4, and this in turn inhibits HRT-DRB4 interaction. These data together with differential localization of DRB4 in plants inoculated with avirulent and virulent viruses, suggests that sub-cellular compartmentalization of DRB4 plays an important role in activation of HRT.

SAG101 forms a ternary complex with EDS1 and PAD4 and is required for resistance signaling against turnip crinkle virus.

EDS1, PAD4, and SAG101 are common regulators of plant immunity against many pathogens. EDS1 interacts with both PAD4 and SAG101 but direct interaction between PAD4 and SAG101 has not been detected, leading to the suggestion that the EDS1-PAD4 and EDS1-SAG101 complexes are distinct. We show that EDS1, PAD4, and SAG101 are present in a single complex in planta. While this complex is preferentially nuclear localized, it can be redirected to the cytoplasm in the presence of an extranuclear form of EDS1. PAD4 and SAG101 can in turn, regulate the subcellular localization of EDS1. We also show that the Arabidopsis genome encodes two functionally redundant isoforms of EDS1, either of which can form ternary complexes with PAD4 and SAG101. Simultaneous mutations in both EDS1 isoforms are essential to abrogate resistance (R) protein-mediated defense against turnip crinkle virus (TCV) as well as avrRps4 expressing Pseudomonas syringae. Interestingly, unlike its function as a PAD4 substitute in bacterial resistance, SAG101 is required for R-mediated resistance to TCV, thus implicating a role for the ternary complex in this defense response. However, only EDS1 is required for HRT-mediated HR to TCV, while only PAD4 is required for SA-dependent induction of HRT. Together, these results suggest that EDS1, PAD4 and SAG101 also perform independent functions in HRT-mediated resistance.

An antiviral defense role of AGO2 in plants.

BACKGROUND: Argonaute (AGO) proteins bind to small-interfering (si)RNAs and micro (mi)RNAs to target RNA silencing against viruses, transgenes and in regulation of mRNAs. Plants encode multiple AGO proteins but, in Arabidopsis, only AGO1 is known to have an antiviral role. METHODOLOGY/PRINCIPAL FINDINGS: To uncover the roles of specific AGOs in limiting virus accumulation we inoculated turnip crinkle virus (TCV) to Arabidopsis plants that were mutant for each of the ten AGO genes. The viral symptoms on most of the plants were the same as on wild type plants although the ago2 mutants were markedly hyper-susceptible to this virus. ago2 plants were also hyper-susceptible to cucumber mosaic virus (CMV), confirming that the antiviral role of AGO2 is not specific to a single virus. For both viruses, this phenotype was associated with transient increase in virus accumulation. In wild type plants the AGO2 protein was induced by TCV and CMV infection. CONCLUSIONS/SIGNIFICANCE: Based on these results we propose that there are multiple layers to RNA-mediated defense and counter-defense in the interactions between plants and their viruses. AGO1 represents a first layer. With some viruses, including TCV and CMV, this layer is overcome by viral suppressors of silencing that can target AGO1 and a second layer involving AGO2 limits virus accumulation. The second layer is activated when the first layer is suppressed because AGO2 is repressed by AGO1 via miR403. The activation of the second layer is therefore a direct consequence of the loss of the first layer of defense.

Two plant viral suppressors of silencing require the ethylene-inducible host transcription factor RAV2 to block RNA silencing.

RNA silencing is a highly conserved pathway in the network of interconnected defense responses that are activated during viral infection. As a counter-defense, many plant viruses encode proteins that block silencing, often also interfering with endogenous small RNA pathways. However, the mechanism of action of viral suppressors is not well understood and the role of host factors in the process is just beginning to emerge. Here we report that the ethylene-inducible transcription factor RAV2 is required for suppression of RNA silencing by two unrelated plant viral proteins, potyvirus HC-Pro and carmovirus P38. Using a hairpin transgene silencing system, we find that both viral suppressors require RAV2 to block the activity of primary siRNAs, whereas suppression of transitive silencing is RAV2-independent. RAV2 is also required for many HC-Pro-mediated morphological anomalies in transgenic plants, but not for the associated defects in the microRNA pathway. Whole genome tiling microarray experiments demonstrate that expression of genes known to be required for silencing is unchanged in HC-Pro plants, whereas a striking number of genes involved in other biotic and abiotic stress responses are induced, many in a RAV2-dependent manner. Among the genes that require RAV2 for induction by HC-Pro are FRY1 and CML38, genes implicated as endogenous suppressors of silencing. These findings raise the intriguing possibility that HC-Pro-suppression of silencing is not caused by decreased expression of genes that are required for silencing, but instead, by induction of stress and defense responses, some components of which interfere with antiviral silencing. Furthermore, the observation that two unrelated viral suppressors require the activity of the same factor to block silencing suggests that RAV2 represents a control point that can be readily subverted by viruses to block antiviral silencing.

Detection of melon necrotic spot virus in water samples and melon plants by molecular methods.

Melon necrotic spot virus (MNSV) is a water and soil-borne pathogen affecting species of the Cucurbitaceae family both in hydroponic and soil crops. Molecular methods for detecting MNSV in water samples, nutrient solutions and melon plants were developed. For this purpose, water samples from a water source pool of a hydroponic culture or from the recirculating nutrient solution were concentrated by ultracentrifugation or PEG precipitation followed by RT-PCR analysis. Both concentration methods were suitable to allow the detection of MNSV and represent, as far as we know, the first time that this virus has been detected in water samples. A non-isotopic riboprobe specific for MNSV was obtained and used to detect the virus in plant tissue. Different parts of mechanically infected plants were examined including the roots, stems, inoculated cotyledons and young leaves. Excluding the inoculated cotyledons, the tissues showing the highest accumulation levels of the virus were the roots. The potential inclusion of such tools in management programs is discussed.