The nuclear localized C2 protein of the monopartite begomovirus Tomato yellow leaf curl virus-China (TYLCV-C) contributes to viral pathogenicity. Here, we have investigated TYLCV-C C2 protein domains that play a role in the phenotype. Alignment of the C2 protein with 67 homologues from monopartite and bipartite begomoviruses revealed that a putative zinc-finger motif C36-X1-C38-X7-C46-X6-H53-X4-H58C59 and four potential phosphorylation sites (T52, S61, Y68, and S74) are highly conserved. When expressed from a Potato virus X (PVX) vector, TYLCV-C C2 protein mutants C2-T52M, C2-H58S, C2-C59S, C2-S61R, and C2-S74D, like the wild-type C2 protein, induced local necrotic ringspots and systemic necrosis in Nicotiana benthamiana plants. Mutants C2-H53P and C2-Y68D produced irregular necrotic lesions on inoculated leaves that were distinct from the wild-type phenotype. In contrast, mutants C2-C36R, C2-C38N, and C2-C46I induced chlorosis and mosaic symptoms rather than necrosis. We demonstrate that TYLCV-C C2, like its counterpart in the bipartite begomovirus African cassava mosaic virus, mediates suppression of posttranscriptional gene silencing (PTGS). Moreover, the individual mutations C36R, C38N, and C46I abolished the ability of C2 protein to suppress PTGS. These results suggest that the three cysteine residues within the putative zinc-finger motif are essential for C2 protein to induce necrosis and to act as a suppressor of PTGS.

The nucleus-localized C2 protein of Tomato yellow leaf curl virus-China (TYLCV-C) is an active suppressor of posttranscriptional gene silencing (PTGS). Consistently, infection with TYLCV-C resulted in PTGS arrest in plants. The C2 protein possesses a functional, arginine-rich nuclear localization signal within the basic amino acid-rich region 17 KVQHRIAKKTTRRRR 31 . When expressed from potato virus X, C2-RRRR 31 DVGG (in which the four consecutive arginine residues 28 RRRR 31 were replaced with DVGG) that had been tagged with a green fluorescent protein (GFP) failed to transport GFP into nuclei and was dysfunctional in inducing necrosis and suppressing PTGS in plants. Amino acid substitution mutants C2-K 17 D-GFP, C2-HR 21 DV-GFP, and C2-KK 25 DI-GFP localized to nuclei and produced necrosis, but only C2-K 17 D-GFP suppressed PTGS. The N-terminal portions C2 1-31 and C2 17-31 fused in frame to GFP were capable of targeting GFP to nuclei, but neither caused necrosis nor affected PTGS. Our data establish that nuclear localization is likely required for C2 protein to function in C2-mediated induction of necrosis and suppression of PTGS, which may follow diverse pathways in plants. Possible mechanisms of how the C2 protein involves these biological functions are discussed.Posttranscriptional gene silencing (PTGS), RNA interference, and gene quelling represent a conserved cellular defense system for controlling foreign gene expression across the plant, animal, and fungal kingdoms (2,6,8,17,32,42,45). These silencing systems involve double-stranded RNA (dsRNA) from which a 21-to 26-nucleotide (nt) short interfering RNA (siRNA) is derived by the action of an RNase III-like dicer, and they share a common molecular mechanism in which a target RNA is degraded in an RNA homology-dependent manner by an RNA-induced silencing multisubunit RNase complex under the guidance of siRNA (2,3,(15)(16)(17)32). In plants, PTGS defends the host against virus infection, down-regulates transgene expression, and may also participate in the control of development (42,45). To counterattack, plant viruses have evolved the ability to encode proteins (i.e., PTGS suppressors) capable of suppressing PTGS by targeting various stages of the PTGS process, including initiation, propagation, and maintenance (6,29,30,42,45). For example, the potyvirus protein HC-Pro affects PTGS maintenance by interfering with a step coincident with, or upstream of, the production of siRNAs (25,27). HC-Pro interacts with a calmodulin-related protein that can suppress PTGS in plants (1). However, the p25 cell-to-cell movement protein of Potato virus X (PVX) and the 2b protein of Cucumber mosaic virus (CMV) preclude the spread of silencing signals (13, 43). On the other hand, the viral PTGS suppressors are often found to be pathogenicity determinants, and their PTGS suppression activity is associated with pathogenicity determination (44). It is worth noting that certain mutants of the CMV 2b protein have been reported to be functional in pathogenesis but dysfunctional in...

Glomerella leaf spot (GLS) caused by Glomerella cingulata is a newly emergent disease that results in severe defoliation and fruit spots. Currently, GLS is not effectively controlled in China due to a lack of understanding of its epidemiology. Therefore, the effects of temperature, wetness duration, and moisture on conidial germination, infection, and the disease incubation period of GLS were examined by inoculating cv. Gala apple leaves with a conidial suspension and performing in vitro germination assays. Conidia could germinate and form appressoria at temperatures ranging from 5 to 35°C, with an optimum temperature of 27.6°C. The germination of conidia required free water or a nearly saturated relative humidity, with only a few conidia germinating and forming appressoria when the RH was less than 99%. The conidial germination dynamics at 10, 25, and 30°C were well represented by three logistic models. The infection of cv. Gala apple leaves by conidia occurred at temperatures ranging from 15 to 35°C. The minimum wetness duration required for infection by conidia at different temperatures was described using a polynomial equation, and the lowest minimum wetness duration was 2.76 h, which occurred at 27.6°C according to the polynomial. Successful infection by conidia was represented by the number of lesions per leaf, which increased with extended wetness durations at the conidial infection stage for six tested temperatures, with the exception of 10°C, when the minimum wetness durations were satisfied. The associations of successfully infected conidia with wetness duration at temperatures of 15, 20, 25, and 30°C were described by four logistic models. Conidia infections developed into visible lesions at temperatures ranging from 15 to 30°C, and the shortest incubation period of 2 days was observed at 25°C. These data and models can be used to construct forecasting models and develop effective control systems for Glomerella leaf spot.

SUMMARY The replication-associated protein (Rep) of two distinct begomoviruses, the bipartite African cassava mosaic virus (ACMV) and the monopartite Tomato yellow leaf curl virus-China (TYLCV-C), elicits a reaction resembling a hypersensitive response (HR), associated with the induction of local necrosis and a systemic burst of hydrogen peroxide production, when expressed from a potato virus X vector in Nicotiana benthamiana. Transient expression of the ACMV Rep after Agrobacterium infiltration of N. benthamiana also triggered an HR-like response. We have identified a region of the ACMV Rep, referred to as the HR-like determinant domain (HRD, amino acids 119-179) that is essential for induction of the phenotype. Two additional regions have been identified (amino acids 1-85 and 86-118) that have various effects on the Rep-mediated phenotype, suggesting that structural constraints are imposed on the functional HRD. The co-expression of Rep with either AC4 or C4, expressed from overlapping open reading frames, triggers systemic necrosis in infected-tissues, but AC4 or C4 alone is neither an inducer nor enhancer of the HR-like phenotype. We propose that ACMV AC4 and TYLCV-C C4 may counter the plant defence mechanism that is initiated by the Rep-mediated local HR-like phenotype.