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P0 protein of some polerovirus members can target ARGONAUTE1 (AGO1) to suppress RNA silencing. Although P0 harbors an F-box-like motif reported to be essential for interaction with S phase kinase-associated protein 1 (SKP1) and RNA silencing suppression, it is the autophagy pathway that was shown to contribute to AGO1 degradation. Therefore, the role of P0-SKP1 interaction in silencing suppression remains unclear. We conducted global mutagenesis and comparative functional analysis of P0 encoded by Brassica yellows virus (BrYV) (P0Br ). We found that several residues within P0Br are required for local and systemic silencing suppression activities. Remarkably, the F-box-like motif mutant of P0Br , which failed to interact with SKP1, is destabilized in vivo. Both the 26S proteasome system and autophagy pathway play a role in destabilization of the mutant protein. Furthermore, silencing of a Nicotiana benthamiana SKP1 ortholog leads to the destabilization of P0Br . Genetic analyses indicated that the P0Br -SKP1 interaction is not directly required for silencing suppression activity of P0Br , but it facilitates stability of P0Br to ensure efficient RNA silencing suppression. Consistent with these findings, efficient systemic infection of BrYV requires P0Br . Our results reveal a novel strategy used by BrYV for facilitating viral suppressors of RNA silencing stability against degradation by plant cells.

Vicinal oxygen chelate (VOC) proteins are members of an enzyme superfamily with dioxygenase or non‐dioxygenase activities. However, the biological functions of VOC proteins in plants are poorly understood. Here, we show that a VOC in Nicotiana benthamiana (NbVOC1) facilitates viral infection. NbVOC1 was significantly induced by infection by beet necrotic yellow vein virus (BNYVV). Transient overexpression of NbVOC1 or its homolog from Beta vulgaris (BvVOC1) enhanced BNYVV infection in N. benthamiana, which required the nuclear localization of VOC1. Consistent with this result, overexpressing NbVOC1 facilitated BNYVV infection, whereas, knockdown and knockout of NbVOC1 inhibited BNYVV infection in transgenic N. benthamiana plants. NbVOC1 interacts with the basic leucine zipper transcription factors bZIP17/28, which enhances their self‐interaction and DNA binding to the promoters of unfolded protein response (UPR)‐related genes. We propose that bZIP17/28 directly binds to the NbVOC1 promoter and induces its transcription, forming a positive feedback loop to induce the UPR and facilitating BNYVV infection. Collectively, our results demonstrate that NbVOC1 positively regulates the UPR that enhances viral infection in plants. [ABSTRACT FROM AUTHOR]

The barley stripe mosaic virus (BSMV) triple gene block 1 (TGB1) protein is required for virus cell-to-cell movement. However, little information is available about how these activities are regulated by post-translational modifications. In this study, we showed that the BSMV Xinjiang strain TGB1 (XJTGB1) is phosphorylated in vivo and in vitro by protein kinase CK2 from barley and Nicotiana benthamiana. Liquid chromatography tandem mass spectrometry analysis and in vitro phosphorylation assays demonstrated that Thr-401 is the major phosphorylation site of the XJTGB1 protein, and suggested that a Thr-395 kinase docking site supports Thr-401 phosphorylation. Substitution of Thr-395 with alanine (T395A) only moderately impaired virus cell-to-cell movement and systemic infection. In contrast, the Thr-401 alanine (T401A) virus mutant was unable to systemically infect N. benthamiana but had only minor effects in monocot hosts. Substitution of Thr-395 or Thr-401 with aspartic acid interfered with monocot and dicot cell-to-cell movement and the plants failed to develop systemic infections. However, virus derivatives with single glutamic acid substitutions at Thr-395 and Thr-401 developed nearly normal systemic infections in the monocot hosts but were unable to infect N. benthamiana systemically, and none of the double mutants was able to infect dicot and monocot hosts. The mutant XJTGB1T395A/T401A weakened in vitro interactions between XJTGB1 and XJTGB3 proteins but had little effect on XJTGB1 RNA-binding ability. Taken together, our results support a critical role of CK2 phosphorylation in the movement of BSMV in monocots and dicots, and provide new insights into the roles of phosphorylation in TGB protein functions.

In the present study, sixteen novel RNA mycoviruses co-infecting a single strain of Rhizoctonia zeae (strain D40) were identified and molecularly characterized using metatranscriptome sequencing combined with a method for rapid amplification of cDNA ends. The fungal strain was isolated from diseased seedlings of sugar beet with damping-off symptoms. Based on genome analysis and phylogenetic analysis of amino acid sequences of RNA-dependent RNA polymerase, the sixteen mycoviruses associated with strain D40 contained three genome types with nine distinct lineages, including positive single-stranded RNA (Hypoviridae, Yadokariviridae, Botourmiaviridae, and Gammaflexiviridae), double-stranded RNA (Phlegiviridae, Megabirnaviridae, Megatotiviridae, and Yadonushiviridae), and negative single-stranded RNA (Tulasviridae), suggesting a complex composition of a mycoviral community in this single strain of R. zeae (strain D40). Full genome sequences of six novel mycoviruses and the nearly full-length sequences of the remaining ten novel mycoviruses were obtained. Furthermore, seven of these sixteen mycoviruses were confirmed to assemble virus particles present in the R. zeae strain D40. To the best of our knowledge, this is the first detailed study of mycoviruses infecting R. zeae. [ABSTRACT FROM AUTHOR]

Abstract Background Beet Necrotic Yellow Vein virus (BNYVV) is a member of the genus Benyvirus causing a worldwide sugar beet disease rhizomania. BNYVV contains four or five plus-sense single stranded RNAs. In altered selective conditions, multipartite RNA viruses of plant are prone to undergoing internal deletions, thus turning into Defective RNAs (D RNAs). Although several D RNAs have been reported in BNYVV infection, the spontaneous internal deletion mutants responsible for severe symptom in systemic host Nicotiana benthamiana (N. benthamiana) are not described so far. Results Systemic host N. benthamiana was inoculated by Chinese BNYVV isolates. RT-PCR and Northern blot showed that the D RNAs forms of BNYVV RNA3 were present in the systemic infection of the N. benthamiana. Three distinct D-RNA3s, named as D-RNA 3α, D-RNA 3β and D-RNA 3γ, were made into infectious clones. When inoculated on the N. benthamiana, the in vitro transcripts of D forms exhibited more stable than that of wild-type RNA3 in systemic movement. Among the detected mutant, the p25 protein frame-shift mutant (D-RNA3α) induced obvious necrotic lesions on Tetragonia.expansa (T. expansa) and pronounced systemic symptom on the N. benthamiana. The D-RNA3α was further mutated artificially to pre-terminate the downstream N protein, leading to the abolishment of the pathogenicity, indicating the N protein was responsible for the necrotic symptom. Conclusion Our studies demonstrated the internal deletion mutants of BNYVV-RNA3 were spontaneously generated in the systemic infection on N. benthamiana. The internal deletions didn't affect the efficient replication of D-RNA3s, instead by improving the stability and pathogenicity of RNA3 in the systemic host N. benthamiana. Besides, our results also suggested the downstream N protein of RNA3, but not the upstream p25 protein, may play an important role in the systemic infection on N. benthamiana.

Barley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.

Cercospora leaf spot (CLS) caused by Cercospora beticola is the most destructive foliar disease of sugar beet worldwide and is mainly controlled by timely fungicide applications. Recently, CLS control by pyraclostrobin exhibited significantly reduced efficiency in some fields of the Chifeng area in Inner Mongolia Autonomous Region of China. Purification of fungi by single spore isolation is essential for the study of fungal and fungicide resistance. However, the conventional single spore isolation and fungicide sensitivity assay for C. beticola are time‐consuming with a risk of contamination. We developed a new method for single spore isolation of C. beticola, which facilitated monitoring fungicide sensitivity of infected field samples. First, the composite spore suspension was prepared from typical lesions by vortex in the sterilized centrifuge tube. Then, the diluted spore suspension was spread on the streptomycin‐amended potato dextrose agar (PDA) plate or the PDA plate contained a threshold dosage of specific fungicide. The C. beticola colony derived from a single spore could be observed with naked eye and easily isolated by a sterilized scalpel knife after 36 h of incubation at 25°C. Meanwhile, the percentage of fungicide‐resistant or ‐tolerant isolates could be analysed based on the growth of different PDA media within 3 days. Based on this method, we identified that 22.88% of the C. beticola isolates were resistant to pyraclostrobin in the Chifeng area, indicating a high risk of emergence of C. beticola‐resistant strains to QoI fungicides in this area. Furthermore, the G143A or F129L mutation of the cytochrome b (Cyt‐b) gene was found in these resistant isolates. To our knowledge, this is the first report of C. beticola QoI‐resistant isolates with G143A or F129L mutation of CbCyt‐b gene in China. Our approach provides considerable potential for studying the biology, fitness and fungicide resistance of C. beticola. [ABSTRACT FROM AUTHOR]