Zhenyue Wang

One-step reverse transcription loop mediated isothermal amplification assay for sensitive and rapid detection of Cucurbit chlorotic yellows virus.

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of Cucurbit chlorotic yellows virus (CCYV). In this procedure, a set of four primers matching a total of six sequences in the coat protein gene region of CCYV was synthesized for the RT-LAMP assay using total RNA extracted from CCYV-infected melon leaf tissues, and the optimum reaction temperature and assay time were determined. The sensitivity assay showed that the virus was detectable in RT-LAMP reactions at dilutions of 1×10(-11), which was 10(5) times more sensitive than the RT-PCR assay. The RT-LAMP assay for CCYV and Sweet potato chlorotic stunt virus (SPCSV) exhibited high specificity for CCYV. This simple and sensitive method has potential for detection of CCYV in samples collected in the field.

Infectious clones of the crinivirus cucurbit chlorotic yellows virus are competent for plant systemic infection and vector transmission.

Cucurbit chlorotic yellows virus (CCYV), a recently identified bipartite crinivirus, causes economic losses in cucurbit plants. CCYV is naturally transmitted only by whitefly Bemisia tabaci. Here we constructed full-length cDNA clones of CCYV (RNA1 and RNA2) fused to the T7 RNA polymerase promoter and the cauliflower mosaic virus 35S promoter. CCYV replicated and accumulated efficiently in Cucumis sativus protoplasts transfected with in vitro transcripts. Without RNA2, RNA1 replicated efficiently in C. sativus protoplasts. Agroinoculation with the infectious cDNA clones of CCYV resulted in systemic infection in the host plants of C. sativus and Nicotiana benthamiana. Virus derived from the infectious clones could be transmitted between cucumber plants by vector whiteflies. This system will greatly enhance the reverse genetic studies of CCYV gene functions.

Two proteins of Cucurbit chlorotic yellows virus, P59 and P9, are self-interacting

Abstract The yeast two-hybrid (Y2H) assay, a powerful tool for identifying protein–protein interactions, has been widely used to study viral protein interactions and to elucidate the functions of viral proteins. In this study, Cucurbit chlorotic yellows virus-encoded proteins were investigated by Y2H assays in all possible pairwise combinations, and the self-interactions of P59 and P9 were detected. The interacting domains of P59 and P9 were identified using vectors carrying an activation domain fused to a truncated version of P59 or P9. We found that the middle region (amino acids 173–344) of P59 was necessary for this self-interaction, while three different truncated versions of P9 showed no interaction with full-length P9. This is the first report of the self-interaction of P59 in the genus Crinivirus.

Transcriptome analysis of Cucumis sativus infected by Cucurbit chlorotic yellows virus

BackgroundCucurbit chlorotic yellows virus (CCYV) is a recently reported bipartite crinivirus that causes chlorotic leaf spots and yellowing symptoms on the leaves of cucurbit plants. The virus–host interaction of CCYV remains to be elucidated, and the influence of criniviruses on the host gene transcriptome requires analysis.MethodsWe used transcriptome sequencing to analyse the differentially expressed genes (DEGs) caused by CCYV infection.ResultsCCYV infection resulted in 865 DEGs. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified 67 pathways, and the three major enrichment pathways (according to the P-values) were photosynthesis-antenna proteins (KO00196), phenylalanine metabolism (KO00360a), and phenylpropanoid biosynthesis (KO00940). Of the 13 DEGs identified in phenylalanine metabolism, 11 genes encode disease resistance-related phenylalanine ammonia-lyase (PAL) genes. Using quantitative real-time PCR, we validated the differential expression of 12 genes.ConclusionsOur study based on the CCYV–cucumber interaction provides comprehensive transcriptomic information, and will improve our understanding of host–crinivirus interactions.

Development of a GFP expression vector for Cucurbit chlorotic yellows virus

BackgroundCucurbit chlorotic yellows virus (CCYV), a bipartite crinivirus, causes chlorotic leaf spots and yellowing symptoms on cucurbit leaves. We previously developed an infectious clone of CCYV. Limited work has been conducted on the construction of a crinivirus green fluorescence protein (GFP) expression vector to date.FindingWe constructed a CCYV GFP expression vector using the “add a gene” strategy based on CCYV RNA2 cDNA constrcut. Three resultant clones, pCCYVGFPSGC, pCCYVGFPCGC, and pCCYVGFPCGS, were constructed with different promoters used to initiate GFP and CP expression. At 25 dpi GFP fluorescence was detectable not only in leaf veins but also in the surrounding cells. pCCYVGFPCGC-infected cucumber leaves exhibited cell spread at 25 dpi, whereas pCCYVGFPSGC and pCCYVGFPCGS were mainly found in single cells. Further observation of pCCYVGFPCGC GFP expression at 30 dpi, 40 dpi, and 50 dpi showed phloem-limited localization in the systemic leaves.ConclusionsWe developed of a CCYV GFP expression vector that will be useful for further study of CCYV movement in cucurbits.

Transgenic Nicotiana benthamiana plants expressing a hairpin RNAi construct of a nematode Rs-cps gene exhibit enhanced resistance to Radopholus similis

Burrowing nematodes (Radopholus similis) cause severe harm in many agronomic and horticultural crops and are very difficult to manage. Cathepsin S is one of the most important cysteine proteinases and plays key roles in nematodes and many other parasites. To evaluate the effect of in planta RNAi on the control of this nematode, a specific fragment from the protease gene, cathepsin S (Rs-cps), was cloned into the binary vector pFGC5941 in the forward and reverse orientations to construct recombinant plant RNAi vectors. Transgenic Nicotiana benthamiana plants expressing Rs-cps dsRNA were obtained and studied. The transcript abundance of Rs-cps dsRNA appeared to be diverse in the different transgenic lines. Moreover, the bioassay results revealed that Rs-cps transgenic N. benthamiana plants were resistant to R. similis and the transcription level of Rs-cps in R. similis was drastically decreased. In addition, the reproduction and hatching rate of R. similis isolated from the Rs-cps transgenic plants were also significantly reduced. Our results suggest that Rs-cps is essential for the reproduction and pathogenicity of R. similis. This is the first study to employ in planta RNAi approach to target the Rs-cps gene for the control of plant parasitic nematodes.

First Report of the Lesion Nematode, Pratylenchus agilis, Parasitizing Wheat in China

Wheat (Triticum aestivum L.) fields in Zhengzhou, Henan Province that exhibited reduced height and number of tillers were surveyed in 2008, and samples were collected for analyses. Fifteen of thirty-five samples contained a nematode suspected of causing small, brown lesions in roots. Both lateral roots and root hairs were reduced in infected plants. Nematode presence was associated with enlarging lesions and necrotic roots. We also observed lesion nematode infestation in samples with no symptoms in the roots. During the growing season, wheat yield increased by 14% with nematocide treatments in Zhengzhou suburb fields. The morphological and molecular analyses of the nematodes isolated from soil samples established the identity of the species as the lesion nematode, Pratylenchus agilis (2,3). Morphological characteristics that were used for identification included female body, lip annules, tail terminus, number of lines in the lateral field, stylet knobs, stylet length, and vulva location in relation to body length. Females were cylindrical, measured 452 to 811 μm long, and contained two lip annules, a smooth tail terminus, four lateral lines, a vulva at 72.64 to 79.97%, a stylet of 16.20 to 17.55 μm, basal esophageal lobes elongated less than twice the body width, and stylet knobs rounded posteriorly. Males were rare and the spermatheca was empty. Molecular analysis was conducted by amplifying the internal transcribed spacer (ITS) regions ITS1 and 2. Sequence of ITS regions from this population (GenBank Accession No. JQ039330) showed highest sequence homology to P. agilis isolate PagKL5 (GenBank Accession No. FJ71289.1) with the identity of 90%. High variability at species level has been found for Pratylenchus species (1). On the basis of the morphological traits and molecular analyses, the nematodes were identified as P. agilis. To our knowledge, this is the first report of P. agilis parasitizing wheat. References: (1) F. De Luca et al. Eur. J. Plant Pathol. 130:415, 2011. (2) A. M. Golden et al. Plant Dis. Rep. 62:430, 1978. (3) R. V. Rebois et al. J. Nematol. 18:392, 1986.