Eureka lemon zinc finger protein ClDOF3.4 interacts with citrus yellow vein clearing virus coat protein to inhibit viral infection

Citrus yellow vein clearing virus(CYVCV)is a new citrus virus that has become an important factor restricting the development of China’s citrus industry,and the CYVCV coat protein(CP)is associated with viral pathogenicity.In this study,the Eureka lemon zinc finger protein(ZFP)ClDOF3.4 was shown to interact with CYVCV CP in vivo and in vitro.Transient expression of ClDOF3.4 in Eureka lemon induced the expression of salicylic acid(SA)-related and hypersensitive response marker genes,and triggered a reactive oxygen species burst,ion leakage necrosis,and the accumulation of free SA.Furthermore,the CYVCV titer in ClDOF3.4 transgenic Eureka lemon plants was approximately 69.4%that in control plants 6 mon after inoculation,with only mild leaf chlorotic spots observed in those transgenic plants.Taken together,the results indicate that ClDOF3.4 not only interacts with CP but also induces an immune response in Eureka lemon by inducing the SA pathways.This is the first report that ZFP is involved in the immune response of a citrus viral disease,which provides a basis for further study of the molecular mechanism of CYVCV infection.

Monoclonal antibody-based serological detection of Citrus yellow vein clearing virus in citrus groves

Citrus yellow vein clearing virus （CYVCV） is considered as the causal agent of Citrus yellow vein clearing disease and belongs to the genus Mandarivirus in the family Alphaflexiviridae. Capsid protein （CP） of CYVCV Chongqing isolate （CYVCV- CQ） was produced using a prokaryotic expression system and used as the immunogen for monoclonal antibody （MAb） production. Four highly specific and sensitive murine MAbs and one polyclonal antibody were prepared in this study. Titers of the four MAbs in ascites fluids ranged from 10-6 to 10-7 as determined by indirect enzyme-linked immunosorbent assay （ELISA）. Three serological assays, including dot enzyme-linked immunosorbent assay （dot-ELISA）, tissue blot-ELISA, and double-antibody sandwich （DAS）-ELISA, were developed for quick and reliable detections of CYVCV in citrus samples. The developed dot-ELISA and DAS-ELISA methods could detect CYVCV in the infected citrus leaf crude extracts diluted at 1：2 560 and 1：10 240 （w/v, g mL^-1）, respectively. The detection result of 125 citrus leaf samples collected from citrus groves in Yunnan Province and Chongqing Municipality of China showed that approximately 36% samples were positive for CYVCV. This virus was, however, not＇detected in any sample collected from Zhejiang or Jiangxi Province, China.

Recent Advances on Citrus yellow vein clearing virus in Citrus

Citrus yellow vein clearing virus(CYVCV)is the causal agent of yellow vein clearing disease,a significant and devastating disease of most citrus species including lemon and sour orange.CYVCV,a single-strand positive-sense RNA virus containing six ORFs(Open Reading Frames),represents a new species in the genus Mandarivirus of the Alphaflexiviridae family.The virus can cause particularly serious damage,resulting in reduced tree vigor,lower yields,and decreased marketability of fruit production,and it has been found in India,Turkey,Pakistan,China and Iran.Here we described the geographical distribution of the virus,its transmission mode by grafting,mechanical inoculation,and insects,as well as currently available techniques for detection.In addition,we also discussed practical measures aimed at controlling the disease and provided theoretical guidance to prevent the acquisition and spread of the disease that is a significant step toward ensuring the health of the citrus industry.

Small RNA deep sequencing reveals full-length genome of Citrus yellow vein clearing virus in Chongqing,China

To identity the potential pathogen associated with the yellow vein clearing symptom on lemon trees, the profiles of virus-de- rived small interfering RNAs from citrus samples were obtained and analyzed by deep sequencing method in this study. Twenty-seven contigs almost cover the full length genome of Citrus yellow vein clearing virus （CYVCV） isolate YN were obtained using the small RNA deep sequencing technology. Analysis showed that this isolate CQ shared the highest nucleotide identity with isolate Y1 （JX040635） and YN （KP313242）, both of which belong to the genus Mandarivirus in the family Alphaflexiviridae. Mapping analysis of viral-derived siRNA （vsiRNA） profiles revealed an uneven distribution pattern of their generations along both positive and negative genome strands, and 22- and 21-nt vsiRNAs ranked the majority. BLAST against viroids and other viral databases confirmed that this sample was single-infected only by CYVCV, which indicated that CYVCV was the exact causal agent for the yellow clearing symptom occurring on lemon. This is the first CYVCV isolate detected in Chongqing and the second in China. This result could provide a molecular basis for the investigation of citrus viral diseases to protect citrus health in this region.

Detection of Citrus yellow vein clearing virus by Quantitative Real-time RT-PCR

To develop a rapid and reliable detection method for Citrus yellow vein clearing virus(CYVCV), a quantitative real-time reverse transcriptionpolymerase chain reaction(q RT-PCR) system based on SYBR Green I was established by using a pair of specific primers designed from its conserved coat protein gene. The sensitivity, specificity, and applicability of the system were evaluated accordingly. The results showed that amplicons were produced from CYVCV isolates, whereas no amplicons from non-CYVCV citrus virus samples, including Citrus tristeza virus(CTV) and Citrus tatter leaf virus(CTLV), were obtained. The sensitivity of the q RT-PCR was 100-fold higher than that of conventional RT-PCR. An excellent linear correlation(R2= 0.999) was obtained from two standard curves of c RNA, and the amplification efficiency was 102%. The data from field citrus samples detection showed that the q RT-PCR system could be used in determining the concentration of CYVCV in different citrus species.