The ghr-miR164 and GhNAC100 modulate cotton plant resistance against Verticillium dahlia.

MicroRNAs (miRNAs) participate in plant development and defence through post-transcriptional regulation of the target genes. However, few miRNAs were reported to regulate cotton plant disease resistance. Here, we characterized the cotton miR164-NAC100 module in the later induction stage response of the plant to Verticillium dahliae infection. The results of GUS fusing reporter and transcript identity showed that ghr-miR164 can directly cleave the mRNA of GhNAC100 in the post-transcriptional process. The ghr-miR164 positively regulated the cotton plant resistance to V. dahliae according to analyses of its over-expression and knockdown. In link with results, the knockdown of GhNAC100 increased the plant resistance to V. dahliae. Based on LUC reporter, expression analyses and yeast one-hybrid (Y1H) assays, GhNAC100 bound to the CGTA-box of GhPR3 promoter and repressed its expression, negatively regulating plant disease resistance. These results showed that the ghr-miR164 and GhNAC100 module fine-tunes plant defence through the post-transcriptional regulation, which documented that miRNAs play important roles in plant resistance to vascular disease.

The Cotton miR477-CBP60A Module Participates in Plant Defense Against Verticillium dahlia.

Previous reports have shown that, when Verticillium dahliae localizes at the root surface, many microRNAs (miRNAs) were identified at the early induction stage. Here, we constructed two groups from two timepoints of small RNA (sRNA) in cotton root responses to V. dahliae at the later induction stage, pathogen localizing in the interior of root tissue. We identified 71 known and 378 novel miRNAs from six libraries of the pathogen-induced and the control sRNAs. Combined with degradome and sRNA sequencing, 178 corresponding miRNA target genes were identified, in which 40 target genes from differentially expressed miRNAs were primarily associated with oxidation-reduction and stress responses. More importantly, we characterized the cotton miR477-CBP60A module in the later response of the plant to V. dahliae infection. A ß-glucuronidase fusion reporter and cleavage site analysis showed that ghr-miR477 directly cleaved the messenger RNA of GhCBP60A in the posttranscriptional process. The ghr-miR477-silencing decreased plant resistance to this fungus, while the knockdown of GhCBP60A increased plant resistance, which regulated GhICS1 expression to determine salicylic acid level. Our data documented that numerous later-inducible miRNAs in the plant response to V. dahliae, suggesting that these miRNAs play important roles in plant resistance to vascular disease.

Cotton WATs Modulate SA Biosynthesis and Local Lignin Deposition Participating in Plant Resistance Against Verticillium dahliae.

Verticillium wilt, caused by Verticillium dahliae, seriously limits cotton production. It is difficult to control this pathogen damage mainly due to the complexity of the molecular mechanism of plant resistance to V. dahliae. Here, we identified three homologous cotton Walls Are Thin (WAT) genes, which were designated as GhWAT1, GhWAT2, and GhWAT3. The GhWATs were predominantly expressed in the roots, internodes, and hypocotyls and induced by infection with V. dahliae and treatment with indole-3-acetic acid (IAA) and salicylic acid (SA). GhWAT1-, GhWAT2-, or GhWAT3-silenced plants showed a comparable phenotype and level of resistance with control plants, but simultaneously silenced three GhWATs (GhWAT123-silenced), inhibited plant growth and increased plant resistance to V. dahliae, indicating that these genes were functionally redundant. In the GhWAT123-silenced plants, the expression of SA related genes was significantly upregulated compared with the control, resulting in an increase of SA level. Moreover, the histochemical analysis showed that xylem development was inhibited in GhWAT123-silenced plants compared with the control. However, lignin deposition increased in the xylem of the GhWAT123-silenced plants compared to the control, and there were higher expression levels of lignin synthesis- and lignifications-related genes in the GhWAT123-silenced plants. Collectively, the results showed that GhWATs in triple-silenced plants acts as negative regulators of plant resistance against V. dahliae. The potential mechanism of the WATs functioning in the plant defence can modulate the SA biosynthesis and lignin deposition in the xylem.

Development of hydrophilic interaction chromatography with quadruple time-of-flight mass spectrometry for heparin and low molecular weight heparin disaccharide analysis.

RATIONALE: Heparin and low molecular weight heparin (LMWH) are widely used as clinical anticoagulants. The determination of their composition and structural heterogeneity still challenges analysts. METHODS: Disaccharide compositional analysis, utilizing heparinase-catalyzed depolymerization, is one of the most important ways to evaluate the sequence, structural composition and quality of heparin and LMWH. Hydrophilic interaction chromatography coupled with quadruple time-of-flight mass spectrometry (HILIC/QTOFMS) has been developed to analyze the resulting digestion products. RESULTS: HILIC shows good resolution and excellent MS compatibility. Digestion products of heparin and LMWHs afforded up to 16 compounds that were separated using HILIC and analyzed semi-quantitatively. These included eight common disaccharides, two disaccharides derived from chain termini, three 3-O-sulfo-group-containing tetrasaccharides, along with three linkage region tetrasaccharides and their derivatives. Structures of these digestion products were confirmed by mass spectral analysis. The disaccharide compositions of a heparin, two batches of the LMWH, enoxaparin, and two batches of the LMWH, nadroparin, were compared. In addition to identifying disaccharides, 3-O-sulfo-group-containing tetrasaccharides, linkage region tetrasaccharides were observed having slightly different compositions and contents in these heparin products suggesting that they had been prepared using different starting materials or production processes. CONCLUSIONS: Thus, compositional analysis using HILIC/QTOFMS offers a unique insight into different heparin products.