Barley yellow dwarf virus-GPV (BYDV-GPV) is transmitted by Rhopalosiphum padi and Schizaphis graminum in a persistent nonpropagative manner. To improve our understanding of its transmission mechanism by aphid vectors, we used two approaches, isobaric tags for relative and absolute quantitation (iTRAQ) and yeast two-hybrid (YTH) system, to identify proteins in R. padi that may interact with or direct the spread of BYDV-GPV along the circulative transmission pathway. Thirty-three differential aphid proteins in viruliferous and nonviruliferous insects were identified using iTRAQ coupled to 2DLC-MS/MS. With the yeast two-hybrid system, 25 prey proteins were identified as interacting with the readthrough protein (RTP) and eight with the coat protein (CP), which are encoded by BYDV-GPV. Among the aphid proteins identified, most were involved in primary energy metabolism, synaptic vesicle cycle, the proteasome pathway and the cell cytoskeleton organization pathway. In a systematic comparison of the two methods, we found that the information generated by the two methods was complementary. Taken together, our findings provide useful information on the interactions between BYDV-GPV and its vector R. padi to further our understanding of the mechanisms regulating circulative transmission in aphid vectors.

Numerous virus pathogens are transmitted by specific arthropod vectors. Understanding the mechanism of transmission is a critical step in the epidemiology of plant viruses and is crucial for the development of effective disease control strategies. In this study, we describe the localization and distribution of Wheat dwarf virus (WDV), an economically important and widespread single-stranded DNA virus, in its leafhopper vector, Psammotettix alienus. The results suggest that WDV not only can move to the salivary glands from the anterior and middle midgut via the hemocoel but also can pass directly through the sheath of the filter chamber and be readily transmitted to healthy wheat plants within 5 min of an acquisition access period on infected plants. When a bacterial-expressed recombinant capsid protein (CP) was incubated with the internal organs of leafhoppers, CP-immunoreactive antigens were found at the anterior and middle midgut. Furthermore, when leafhoppers were fed with an antiserum raised against the CP, the accumulation of WDV in the gut cells, hemocoel, and salivary glands was significantly reduced. These data provide evidence that transmission of WDV is determined by a CP-mediated virion–vector retention mechanism.

In May of 2004 and 2005, several diseased wheat (Triticum aestivum L.) plants showing extreme dwarfing, various types of yellowing, and reduced or no heading were found in the breeding fields of the Institute of Crop Science, Shanxi Academy of Agricultural Sciences, Taiyuan, Shanxi Province, China. On the basis of these symptoms, infection with Wheat dwarf virus (WDV) was suspected. Total DNA was extracted from diseased plants with the DNeasy Plant Mini kit (Qiagen, Hilden, Germany). Primers were designed based on the WDV-Enkoping1 genome sequence (NC_003326) (1), including: 245f, 5′-CGACTACGCCTGGCGAACATTTG-3′ (residues 245–267); 806r, 5′-TCTGGCATTGCCTGTTTCGG-3′ (complementary to residues 787–806); 1381f, 5′-CAGTGACATCTTCGCCGGAG-3′ (residues 1381–1400); and, 1886r, 5′-ACTCCGTAAGCCTCGAATCC-3′ (complementary to residues 1867–1886). With primer pairs 245f/806r, 1381f/1886r, 245f/1886r, and 1381f/806r, PCR products of 560, 506, 1642, and 2,275 bp were expected, respectively. After amplification, fragments of the expected sizes were seen on 1% (w/v) agarose gels. The fragments were purified by using a DNA gel extraction kit (TaKaRa, Dalian, China) and cloned into the pGEM-T vector (Promega, Madison, WI). The plasmids were transformed into E. coli strain DH5α and plasmid DNA was isolated from overnight cultures by alkaline lysis. Insert sequences were determined using the dideoxynucleotide chain termination method with an automated sequencer (ABI BigDye 3.1, Applied Biosystems, Foster City, CA). At least three independently isolated clones were analyzed for each PCR product. The compiled 2,750 nt sequence (GenBank Accession No. DQ868525) was 98.1, 98.5, 97.8, and 97.9% identical to WDV-Enkoping1 (NC_003326), WDV-SE (X02869), WDV-B (AM040732), and WDV-F (AM040733), respectively. Therefore, the virus isolate (WDV-TY) was identified as WDV (genus Mastrevirus, family Geminividae). Wheat samples collected from different provinces from 2004-2006 were also infected with WDV as indicated by PCR using the same primer pairs. For Shijiazhuang (Hebei Province), Yangling (Shanaxi Province), Kunming (Yunnan Province), Yuncheng (Shanxi Province), Tianshui (Gansu Province), Gangu (Gansu Province), and Zhenzhou (Henan Province), 13 of 14, 6 of 6, 5 of 5, 4 of 4, 2 of 3, 1 of 2, and 1 of 1 samples were positive, respectively, indicating a broad distribution of WDV in China. To our knowledge, this is the first report of WDV in wheat in China. Reference: (1) A. Kvarnheden et al. Arch Virol. 147:205, 2002.

Several members of the family Luteoviridae are important pathogens of cultivated plant species of the family Gramineae. In this study, we explored RNA-silencing suppressors (RSSs) encoded by two cereal-infecting luteoviruses: barley yellow dwarf virus and wheat yellow dwarf virus (BYDV and WYDV, respectively). The P0 protein of WYDV-GPV (P0^GPV) and the P6 protein of BYDV-GAV (P6^GAV) displayed RSS activities when expressed in agro-infiltrated leaves of Nicotiana benthamiana, by their local ability to inhibit post-transcriptional gene silencing of GFP. Analysis of GFP, mRNA and GFP-specific small interfering RNA indicated that both P0^GPV and P6^GAV are suppressors of silencing that can restrain not only local but also systemic gene silencing. This is the first report of RSS activity of the P6 protein in a member of the genus Luteovirus.

To identify the pathogens responsible for leaf yellowing symptoms on wheat samples collected from Jinan, China, we tested for the presence of three known barley/wheat yellow dwarf viruses (BYDV-GAV, -PAV, WYDV-GPV) (most likely pathogens) using RT-PCR. A sample that tested negative for the three viruses was selected for small RNA sequencing. Twenty-five million sequences were generated, among which 5% were of viral origin. A novel polerovirus was discovered and temporarily named wheat leaf yellowing-associated virus (WLYaV). The full genome of WLYaV corresponds to 5 772 nucleotides (nt), with six AUG-initiated open reading frames, one non-AUG-initiated open reading frame and three untranslated regions, showing typical features of the family Luteoviridae. Sequence comparison and phylogenetic analyses suggested that WLYaV had the closest relationship with sugarcane yellow leaf virus (ScYLV), but the identities of full genomic nucleotides and deduced amino acid sequence of coat protein (CP) were 64.9% and 86.2%, respectively, below the species demarcation thresholds (90%) in the family Luteoviridae. Furthermore, agroinoculation of Nicotiana benthamiana leaves with a cDNA clone of WLYaV caused yellowing symptoms on the plant. Our study adds a new polerovirus that is associated with wheat leaf yellowing disease, which would help to identify and control pathogens of wheat.