The Toll-Spätzle pathway is a crucial defense mechanism in insect innate immunity, it plays an important role in fighting against pathogens through the regulation of antimicrobial peptide gene expression. Although Toll and Spätzle (Spz) genes have been identified in Bombyx mori, little is known regarding the specific Spz and Toll genes members involved in innate immunity. There is also limited direct evidence of the interaction between Spz and Toll. In this study, the dual-luciferase reporter assay results showed that BmToll11 and BmToll9–1 could activate both drosomycin and diptericin promoters in S2 cells. Furthermore, BmToll11, BmToll9–1, and five BmSpzs genes were found to be significantly upregulated in B. mori infected by Escherichia coli and Staphylococcus aureus. Additionally, the yeast two-hybrid assay results confirmed that BmSpz2, but not other BmSpzs, could interact with both BmToll11 and BmToll9–1. These findings suggest that the activated BmSpz2 can bind with BmToll11 and BmToll9–1 to induce the expression of AMPs after the silkworm is infected by pathogens.
A rapid immunochromatographic strip (ICS) test for detection of citrus yellow vein clearing virus (CYVCV) was developed. The test is based on an antibody sandwich format and uses the monoclonal antibody (MAb) 1E1, which is specific for CYVCV. MAb 1E1 labeled with 30-nm colloidal gold particles was coated on a gold conjugate pad. A secondary goat anti-mouse IgG was coated on the surface of a nitrocellulose filter membrane (NC) as the control (C) line, while 1E1 was coated on the surface of the NC as the test (T) line. The ICS test was evaluated for specificity and sensitivity and then applied for virus detection in field samples. There was no cross-reaction with citrus tristeza virus (CTV), satsuma dwarf virus (SDV), citrus tatter leaf virus (CTLV), citrus exocortis viroid (CEVd), citrus mosaic virus (CiMV), citrus psorosis virus (CPV), citrus ringspot virus (RSV) or 'Candidatus Liberibacter asiaticus' (CLas). The ICS test was still able to detect CYVCV in tissue extracts at a dilution of 1: 320 (w/v), which is as efficient as the dot-ELISA assay. In general, the ICS assay is less expensive, faster and simpler to conduct than conventional CYVCV detection methods, so it may be useful for large-scale detection or monitoring of CYVCV.
Citrus yellow vein clearing virus is a new member of the genus Mandarivirus in the family Alphaflexiviridae. Citrus yellow vein clearing virus (CYVCV) is the causal agent of citrus yellow vein clearing disease and is widely distributed in Pakistan, India, Turkey, and China. CYVCV is transmitted from citrus to citrus by Dialeurodes citri, grafting, and contaminated knife blades, threatening citrus production. In this study, four infectious full-length cDNA clones of CYVCV (namely AY112, AY132, AY212, and AY221) derived from CYVCV isolate AY were obtained through yeast homologous recombination and inoculated to ‘Eureka’ lemon (Citrus limon Burm. f.) by Agrobacterium-mediated vacuum infiltration. Pathogenicity analysis indicated that the clones AY212 and AY221 caused more severe symptoms than AY112 and AY132. Northern blot and quantitative reverse transcription PCR (qRT-PCR) analyses showed that the titers of virulent clones (AY212 and AY221) were significantly higher than those of attenuated clones (AY112 and AY132) in the infected ‘Eureka’ lemon (Citrus limon Burm. f.) seedlings. Subsequent comparative studies of viral infectivity, accumulation, and symptoms induced by AY221 in nine citrus cultivars indicated that (i) the infectivity of AY221 varied from 25% to 100% among different cultivars; (ii) ‘Oota’ ponkan (C. reticulata L.) showed the lowest infection rate with mild symptoms, which might be a useful resource for CYVCY-resistance genes; (iii) CYVCV titer was positively associated with the symptom development in infected citrus seedlings. In general, this report revealed the biological properties of CYVCV, thus laying a foundation for further investigation of pathogenic mechanisms in this virus.
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