Changyong Zhou

Satellite RNAs interfere with the function of viral RNA silencing suppressors

Viral satellite RNAs (satRNAs) are small subviral RNAs and depend on the helper virus for replication and spread. satRNAs can attenuate helper virus-induced symptoms, the mechanism of which remains unclear. Here, we show that two virus-encoded suppressors of RNA silencing (VSRs), Cucumber mosaic virus (CMV) 2b and Tombusvirus P19, suppress hairpin RNA (hpRNA)-induced silencing of a β-glucuronidase (GUS) gene in Nicotiana benthamiana. This suppression can be overcome by CMV Y-satellite RNA (Y-Sat) via the Y-Sat-derived small interfering RNAs (siRNAs), which bind to the VSRs and displace the bound hpGUS-derived siRNAs. We also show that microRNA target gene expression in N. tabacum was elevated by CMV infection, presumably due to function of the 2b VSR, but this upregulation of microRNA target genes was reversed in the presence of Y-Sat. These results suggest that satRNA infection minimizes the effect of VSRs on host siRNA and microRNA-directed silencing. Our results suggest that the high abundance of satRNA-derived siRNAs contributes to symptom attenuation by binding helper virus-encoded VSRs, minimizing the capacity of the VSRs to bind host siRNA and miRNA and interfere with their function.

Ultrastructural Changes and Putative Phage Particles Observed in Sweet Orange Leaves Infected with ‘Candidatus Liberibacter asiaticus’

Huanglongbing (HLB), also known as citrus greening, is currently the most destructive citrus disease. Anatomical analyses of HLB-affected sweet orange were carried out by light and electron microscopy. As compared with healthy citrus, the phloem plasmodesmata were plugged with callose, and in some samples the phloem was collapsed. Chloroplast structures were deformed. Prophage sequences occupy a significant portion of the genome of Candidatus Liberibacter asiaticus and have been used to distinguish strains from Yunnan and Guangdong provinces in China and Florida. Interestingly, a large number of possible putative phage particles were observed attached on the surface of Ca. L. asiaticus cells in plants inoculated with strain FJ3 from Fujian Province, China. Phage particles have been observed previously only in periwinkle plants artificially inoculated in Florida with Ca. L. asiaticus that carried the SC1-type prophage. PCR assays verified the presence of the SC1-type prophage sequences previously described from this bacterium in Florida in the FJ3 isolate. This is the first time that suspected phage particles have been observed in sweet orange trees infected with Ca. L. asiaticus.

RT-PCR-RFLP for genetic diversity analysis of the citrus tatter leaf virus strain of Apple stem grooving virus

A method for detecting genetic diversity of the Citrus tatter leaf virus (CTLV) strain of Apple stem grooving virus was developed based on restriction fragment length polymorphisms (RFLP) of the 889xa0nt 3′ sequence amplified by RT-PCR, and nine Hinf I RFLP patterns were defined. This RFLP assay, together with biological indexing and sequencing, was applied to characterize 18 CTLV isolates collected from seven provinces of China. The results indicated that the majority of these isolates (16/18) presented as single well-defined RFLP patterns, among which RFLPIand RFLPII were dominant. In contrast, two samples exhibited as a mixture of two RFLP patterns, suggesting a mixed infection of CTLV variants. Of the 18 isolates, six of eight samples with RFLPIIor RFLP III induced mild to moderate symptoms on indexing plants (Citrus. sinensisu2009×u2009Poncirus trifoliata cv. Rusk), while four isolates conforming to RFLPIand six samples to RFLP IVu2009~u2009IX induced severe symptoms. In the phylogenetic tree constructed according to the 3′ nucleotide sequence of the 18 CTLV isolates, RFLPIIand RFLP III samples clustered into phylogenetic group A, whereas the others gathered into phylogenetic group B. These results revealed correlation among mild (moderate) symptoms, phylogenetic group A and RFLPIIor RFLP III restrictotypes, suggesting the RFLP assay may be useful in quick identification of CTLV strains.

Molecular characterization of Citrus tristeza virus isolates from Pakistan based on CPG/Hinf I restriction fragment length polymorphism (RFLP) groups analysis

From six different districts of Punjab, Pakistan, 85 isolates of Citrus tristeza virus (CTV) were collected and characterized based on coat protein gene (CPG) analysis. All isolates were collected from field trees showing various CTV symptoms such as decline in most citrus varieties, inverse pitting on some sour orange rootstocks below bud union, mild-to-moderate stem-pitting on the trunk of some sweet orange. The CTV CP gene of all isolates was amplified by reverse transcriptase polymerase chain reaction (RT–PCR) using CP gene-specific primers yielding 672 bp. The maximum disease incidence was found in sweet orange followed by mandarin and grapefruit. These isolates were then subjected to CPG/Hinf I restriction fragment length polymorphism (RFLP) analysis. Mixed infection of CTV isolates was found very common in the field tress in Pakistan. The most dominant CPG/Hinf I RFLP groups III, I and VI are the basic causal epidemic in Pakistan. Moreover, based on symptoms in the field trees, CPG/Hinf I RFLP groups III, I and VI are considered to be the obvious causes of decline and stem-pitting in Pakistan. Key words: Citrus tristeza virus, CPG/Hinf I restriction fragment length polymorphism (RFLP) groups, decline, stem-pitting.

Isolation and characterization of Zhihengliuella aestuarii B18 suppressing clubroot on Brassica juncea var. tumida Tsen

Mustard clubroot, caused by Plasmodiophora brassicae, is a serious disease that affects Brassica juncea var. tumida Tsen, a mustard plant that is the raw material for a traditional fermented food manufactured in the Chongqing Municipality, People’s Republic of China. To find antagonistic bacteria for P. brassicae, 124 bacteria were obtained from the rhizosphere soil of B. juncea var. tumida grown in Fuling, Chongqing. Isolates were preliminarily chosen by evaluating the inhibition rate of the P. brassicae resting spore germination. The biocontrol effects of three antagonistic bacteria against clubroot on B. juncea var. tumida were evaluated in a greenhouse experiment. B18 showed the highest control efficiency, at 63.4% in the greenhouse test. In a field trial, B18 was also effective in controlling clubroot, but only at a 49.7% efficiency rate. According to 16S rDNA sequence analysis, strain B18 had a 100% sequence similarity with type strain Zhihengliuella aestuarii DY66T (EU939716). Based on morphological, cultural, physiological and biochemical characteristics, the DNA Gxa0+xa0C content, polar lipids, fatty acids, cell wall analysis, as well as DNA–DNA hybridization, strain B18 was identified as Z. aestuarii B18. Thus, the isolate B18 might have a potential biocontrol application for clubroot. We report for the first time that Z. aestuarii B18 can control clubroot.

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

Abstract To identity the potential pathogen associated with the yellow vein clearing symptom on lemon trees, the profiles of virus-derived 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.

Co-infection of Sweet Orange with Severe and Mild Strains of Citrus tristeza virus Is Overwhelmingly Dominated by the Severe Strain on Both the Transcriptional and Biological Levels

Citrus tristeza is one of the most destructive citrus diseases and is caused by the phloem-restricted Closterovirus, Citrus tristeza virus. Mild strain CTV-B2 does not cause obvious symptoms on indicators whereas severe strain CTV-B6 causes symptoms, including stem pitting, cupping, yellowing, and stiffening of leaves, and vein corking. Our laboratory has previously characterized changes in transcription in sweet orange separately infected with CTV-B2 and CTV-B6. In the present study, transcriptome analysis of Citrus sinensis in response to double infection by CTV-B2 and CTV-B6 was carried out. Four hundred and eleven transcripts were up-regulated and 356 transcripts were down-regulated prior to the onset of symptoms. Repressed genes were overwhelmingly associated with photosynthesis, and carbon and nucleic acid metabolism. Expression of genes related to the glycolytic, oxidative pentose phosphate (OPP), tricarboxylic acid cycle (TCA) pathways, tetrapyrrole synthesis, redox homeostasis, nucleotide metabolism, protein synthesis and post translational protein modification and folding, and cell organization were all reduced. Ribosomal composition was also greatly altered in response to infection by CTV-B2/CTV-B6. Genes that were induced were related to cell wall structure, secondary and hormone metabolism, responses to biotic stress, regulation of transcription, signaling, and secondary metabolism. Transport systems dedicated to metal ions were especially disturbed and ZIPs (Zinc Transporter Precursors) showed different expression patterns in response to co-infection by CTV-B2/CTV-B6 and single infection by CTV-B2. Host plants experienced root decline that may have contributed to Zn, Fe, and other nutrient deficiencies. Though defense responses, such as, strengthening of the cell wall, alteration of hormone metabolism, secondary metabolites, and signaling pathways, were activated, these defense responses did not suppress the spread of the pathogens and the development of symptoms. The mild strain CTV-B2 did not provide a useful level of cross-protection to citrus against the severe strain CTV-B6.

Genome characterization of sweet potato symptomless virus 1: a mastrevirus with an unusual nonanucleotide sequence

Complete genomic sequences of nine isolates of sweet potato symptomless virus 1 (SPSMV-1), a virus of the genus Mastrevirus in the family Geminiviridae, were determined from sweet potato accessions from different countries and found to be 2,559-2,602 nucleotides in length. These isolates shared 97-100% genome sequence identity and had an unusual nonanucleotide sequence (TAAGATTCC) in a large intergenic region as well as an additional open reading frame, C3, which is conserved in dicot-infecting mastreviruses.

The complete genome sequence of Citrus vein enation virus from China

The complete nucleotide sequence of an isolate of Citrus vein enation virus(CVEV-XZG) from China has been determined for the first time. The genome consisted of 5 983 nucleotides, coding for five open reading frames(ORFs), had a similar genomic organization features with Pea enation mosaic virus(PEMV). Nucleotide and deduced amino acid sequence identity of the five ORFs compared to isolate CVEV VE-1 range from 97.1 to 99.0% and 97.4 to 100.0%, these values compared to isolate PEMV-1 range from 45.2 to 51.6% and 31.1 to 45.2%. Phylogenetic analysis based on the complete genome sequence showed that the isolate CVEV-XZG had close relationship with Pea enation mosaic virus. The results supports CVEV may be a new member of genus Enamovirus. The full sequence of CVEV-XZG presented here may serve as a basis for future study of CVEV in China.

Complete genome sequence analysis of two Citrus tatter leaf virus (CTLV) isolates from China

Abstract In order to understand molecular characterization of Citrus tatter leaf virus (CTLV) isolated from China, full-length cDNAs of CTLV-MTH and CTLV-XHC from Citrus reticulata and Citrus sinensis were cloned and sequenced based on whole-genome amplification by RT-PCR. The complete nucleotide sequences of CTLV-MTH and CTLV-XHC were determined to be 6 497 nucleotides in length and shared 79.9–91.0% and 78.8–98.0% nucleotide sequence identity, respectively, with other Apple stem grooving virus (ASGV) or CTLV strains available in GenBank. Unexpectedly, CTLV-MTH showed the highest nucleotide sequence identity (91%) with an apple isolate of ASGV, followed by 86.5% with ASGV-HH and 85.7% with ASGV-CHN. Furthermore, CTLV-MTH and three ASGV strains were grouped to a separate cluster in the phylogenetic tree, suggesting it has a closer relationship to ASGV than to CTLV. Therefore, it can be concluded roughly that CTLV may be not a distinct strains of ASGV. We proposed that Citrus tatter leaf virus should be renamed Apple stem grooving virus.