SUMMARYCucumber mosaic virus suppressor 2b (CMV2b) is a nuclear viral suppressor that interferes with local and systemic silencing and inhibits AGO1 slicer activity. CMV2b-mediated transgene hypomethylation and its localization in Cajal bodies suggests a role of CMV2b in RNA-directed DNA methylation (RdDM). However, its direct involvement in RdDM, or its binding with small RNAs (sRNAs) in vivo is not yet established. Here, we show that CMV2b binds both microRNAs (miRNAs) and small interfering RNAs (siRNAs) in vivo. sRNA sequencing data from the CMV2b immunocomplex revealed its preferential binding with 24-nt repeatassociated siRNAs. We provide evidence that CMV2b also has direct interaction with the AGO4 protein by recognizing its PAZ and PIWI domains. Subsequent analysis of AGO4 functions revealed that CMV2b reduced AGO4 slicer activity and the methylation of several loci, accompanied by the augmented accumulation of 24-nt siRNAs in Arabidopsis inflorescences. Intriguingly, CMV2b also regulated an AGO4-related epiallele independently of its catalytic potential, which further reinforces the repressive effects of CMV2b on AGO4 activity. Collectively, our results demonstrate that CMV2b can counteract AGO4-related functions. We propose that by adopting novel counter-host defense strategies against AGO1 and AGO4 proteins, CMV creates a favorable cellular niche for its proliferation.

SUMMARYNitrate (NO À 3 ) is a key signaling molecule in plant metabolism and development, in addition to its role as a nutrient. It has been shown previously in Arabidopsis that ANR1, a MADS-box transcription factor, is a major component in the NO À 3 -signaling pathway that triggers lateral root growth and that miR444, which is specific to monocots, targets four genes that are homologous to ANR1 in rice. Here, we show that miR444a plays multiple roles in the rice NO À 3 -signaling pathway -not only in root development, but also involving nitrate accumulation and even P i -starvation responses. miR444a overexpression resulted in reduced rice lateral root elongation, but promoted rice primary and adventitious root growth, in a nitrate-dependent manner. In addition, overexpression of miR444a improved nitrate accumulation and expression of nitrate transporter genes under high nitrate concentration conditions, but reduced the remobilization of nitrate from old leaves to young leaves thus affecting the plant's ability to adapt to nitrogen-limitating conditions. Intriguingly, we found that P i starvation strongly induced miR444 accumulation in rice roots and that overexpression of miR444a altered P i -starvation-induced root architecture and enhanced P i accumulation and expression of three P i transporter genes. We further provide evidence that miR444a is involved in the interaction between the NO À 3 -signaling and P i -signaling pathways in rice. Taken together, our observations demonstrated that miR444a plays multiple roles in the rice NO À 3 -signaling pathway in nitrate-dependent root growth, nitrate accumulation and phosphate-starvation responses.

Plant RNA-DEPENDENT RNA POLYMERASE1 (RDR1) is a key component of the antiviral RNA-silencing pathway, contributing to the biogenesis of virus-derived small interfering RNAs. This enzyme also is responsible for producing virusactivated endogenous small interfering RNAs to stimulate the broad-spectrum antiviral activity through silencing host genes. The expression of RDR1 orthologs in various plants is usually induced by virus infection. However, the molecular mechanisms of activation of RDR1 expression in response to virus infection remain unknown. Here, we show that a monocot-specific microRNA, miR444, is a key factor in relaying the antiviral signaling from virus infection to OsRDR1 expression. The expression of miR444 is enhanced by infection with Rice stripe virus (RSV), and overexpression of miR444 improves rice (Oryza sativa) resistance against RSV infection accompanied by the up-regulation of OsRDR1 expression. We further show that three miR444 targets, the MIKC C -type MADS box proteins OsMADS23, OsMADS27a, and OsMADS57, form homodimers and heterodimers between them to repress the expression of OsRDR1 by directly binding to the CArG motifs of its promoter. Consequently, an increased level of miR444 diminishes the repressive roles of OsMADS23, OsMADS27a, and OsMADS57 on OsRDR1 transcription, thus activating the OsRDR1-dependent antiviral RNA-silencing pathway. We also show that overexpression of miR444-resistant OsMADS57 reduced OsRDR1 expression and rice resistance against RSV infection, and knockout of OsRDR1 reduced rice resistance against RSV infection. In conclusion, our results reveal a molecular cascade in the rice antiviral pathway in which miR444 and its MADS box targets directly control OsRDR1 transcription.RNA silencing mediated by regulatory small RNAs (microRNAs [miRNAs] and small interfering RNAs [siRNAs]) negatively regulates gene expression at the posttranscriptional level or at the transcriptional level in eukaryotic organisms. Besides small RNAs, plant RNA-silencing pathways incorporate several kinds of core protein components, such as DICER-LIKE (DCL) RNase III endonucleases, which process long doublestranded RNA (dsRNA) into small RNA duplexes; ARGONAUTEs (AGOs), the major effector of the RNA-induced silencing complexes, which bind to small RNAs for silencing target RNAs; and RNA-dependent RNA polymerases (RDRs), which are required for copying single-stranded RNAs into dsRNAs for downstream processing by DCLs. Multiple DCLs, AGOs, and RDRs have evolved in plants and thus form an array of RNA-silencing pathways (Axtell, 2013;Martínez de Alba et al., 2013;Bologna and Voinnet, 2014). Among them, the antiviral RNA-silencing pathway is the earliest described and most extensively studied. It is well known that the antiviral silencing pathway directly targets viral RNAs. Briefly, as in Arabidopsis (Arabidopsis thaliana), the stem-loop structures and dsRNA replication intermediates of viral RNAs are recognized and cleaved by DCLs (DCL4 and DCL2) to produce primary virus-derived small interferi...

ORCID IDs: 0000-0003-0044-6830 (G.B.M.); 0000-0001-8802-4872 (L.Z.).The activation of an immune response in tomato (Solanum lycopersicum) against Pseudomonas syringae relies on the recognition of E3 ligase-deficient forms of AvrPtoB by the host protein kinase, Fen. To investigate the mechanisms by which Fen-mediated immunity is regulated, we characterize in this study a Fen-interacting protein, Fni3, and its cofactor, S. lycoperiscum Uev (Suv). Fni3 encodes a homolog of the Ubc13-type ubiquitin-conjugating enzyme that catalyzes exclusively Lys-63-linked ubiquitination, whereas Suv is a ubiquitin-conjugating enzyme variant. The C-terminal region of Fen was necessary for interaction with Fni3, and this interaction was required for cell death triggered by overexpression of Fen in Nicotiana benthamiana leaves. Fni3 was shown to be an active E2 enzyme, but Suv displayed no ubiquitin-conjugating activity; Fni3 and Suv together directed Lys-63-linked ubiquitination. Decreased expression of Fni3, another tomato Ubc13 homolog, Sl-Ubc13-2, or Suv in N. benthamiana leaves diminished cell death associated with Fen-mediated immunity and cell death elicited by several other resistance (R) proteins and their cognate effectors. We also discovered that coexpression of Fen and other R proteins/effectors with a Fni3 mutant that is compromised for ubiquitin-conjugating activity diminished the cell death. These results suggest that Fni3/Sl-Ubc13-2 and Suv regulate the immune response mediated by Fen and other R proteins through Lys-63-linked ubiquitination.

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Potato is regarded as drought sensitive and most vulnerable to climate changes. Its cultivation in drought prone regions or under conditions of more frequent drought periods, especially in subtropical areas, requires intensive research to improve drought tolerance in order to guarantee high yields under limited water supplies. A candidate gene approach was used to develop functional simple sequence repeat (SSR) markers for association studies in potato with the aim to enhance breeding for drought tolerance. SSR primer combinations, mostly surrounding interrupted complex and compound repeats, were derived from 103 candidate genes for drought tolerance. Validation of the SSRs was performed in an association panel representing 34 mainly starch potato cultivars. Seventy-five out of 154 SSR primer combinations (49%) resulted in polymorphic, highly reproducible banding patterns with polymorphic information content (PIC) values between 0.11 and 0.90. Five SSR markers identified allelic differences between the potato cultivars that showed significant associations with drought sensitivity. In all cases, the group of drought-sensitive cultivars showed predominantly an additional allele, indicating that selection against these alleles by marker-assisted breeding might confer drought tolerance. Further studies of these differences in the candidate genes will elucidate their role for an improved performance of potatoes under water-limited conditions.

Top-Down Proteomics (TDP) is an emerging proteomics protocol that involves identification, characterization, and quantitation of intact proteins using high-resolution mass spectrometry. TDP has an edge over other proteomics protocols in that it allows for: (i) accurate measurement of intact protein mass, (ii) high sequence coverage, and (iii) enhanced identification of post-translational modifications (PTMs). However, the complexity of TDP spectra poses a significant impediment to protein search and PTM characterization. Furthermore, limited software support is currently available in the form of search algorithms and pipelines. To address this need, we propose ‘SPECTRUM’, an open-architecture and open-source toolbox for TDP data analysis. Its salient features include: (i) MS2-based intact protein mass tuning, (ii) de novo peptide sequence tag analysis, (iii) propensity-driven PTM characterization, (iv) blind PTM search, (v) spectral comparison, (vi) identification of truncated proteins, (vii) multifactorial coefficient-weighted scoring, and (viii) intuitive graphical user interfaces to access the aforementioned functionalities and visualization of results. We have validated SPECTRUM using published datasets and benchmarked it against salient TDP tools. SPECTRUM provides significantly enhanced protein identification rates (91% to 177%) over its contemporaries. SPECTRUM has been implemented in MATLAB, and is freely available along with its source code and documentation at https://github.com/BIRL/SPECTRUM/ .