The draft genome sequence of Xylella fastidiosa subsp. multiplex strain Griffin-1, isolated from a red oak tree (Quercus rubra) in Georgia, is reported here. The bacterium has a genome size of 2,387,314 bp, with a G+C content of 51.7%. The Griffin-1 strain genome contains 2,903 predicted open reading frames and 50 RNA genes.
Replacement of diseased plants with healthy plants is commonly used to manage spread of plant pathogens in perennial cropping systems. This strategy has two potential benefits. First, removing infected plants may slow pathogen spread by eliminating inoculum sources. Second, replacing infected plants with uninfected plants may offset yield losses due to disease. The extent to which these benefits are realized depends on multiple factors. In this study, sensitivity analyses of two spatially explicit simulation models were used to evaluate how assumptions concerning implementation of a plant replacement program and pathogen spread interact to affect disease suppression. In conjunction, effects of assumptions concerning yield loss associated with disease and rates of plant maturity on yields were simultaneously evaluated. The first model was used to evaluate effects of plant replacement on pathogen spread and yield on a single farm, consisting of a perennial crop monoculture. The second model evaluated effects of plant replacement on pathogen spread and yield in a 100 farm crop growing region, with all farms maintaining a monoculture of the same perennial crop. Results indicated that efficient replacement of infected plants combined with a high degree of compliance among farms effectively slowed pathogen spread, resulting in replacement of few plants and high yields. In contrast, inefficient replacement of infected plants or limited compliance among farms failed to slow pathogen spread, resulting in replacement of large numbers of plants (on farms practicing replacement) with little yield benefit. Replacement of infected plants always increased yields relative to simulations without plant replacement provided that infected plants produced no useable yield. However, if infected plants produced useable yields, inefficient removal of infected plants resulted in lower yields relative to simulations without plant replacement for perennial crops with long maturation periods in some cases.
Circulifer tenellus virus 1 (CiTV1) is the prototypical example of an unusual group of dsRNA viruses associated with insects and for which ecological data are lacking. A San Joaquin Valley (SJV), California population of the beet leafhopper (BLH; Circulifer tenellus [Baker]) was sampled for CiTV1 in 2010. Among 365 BLH sampled, 119 (32.6%) were positive for CiTV1, with at least one CiTV1-positive BLH collected from each of 35 locations. Chi-square tests indicated that observed CiTV1 incidence differed from expected values based on collection season but not geography within the SJV. Sequence comparisons identified three CiTV1 strains, designated A, B, and C. Strain A predominated (82.4%), strain B was less common (16.8%), and only one (0.8%) strain C isolate was encountered. Chi-square tests demonstrated that observed frequencies of strains A and B did not differ from expected values in space or time, indicating that the SJV population of CiTV1 was unstructured.
Next generation sequence analyses were used to assess virus-derived small RNA (vsRNA) profiles for Homalodisca coagulata virus-1 (HoCV-1), family Dicistroviridae, and Homalodisca vitripennis reovirus (HoVRV), family Reoviridae, from virus-infected H. vitripennis, the glassy-winged sharpshooter. The vsRNA reads were mapped against the monopartite genome of HoCV-1 and all 12 genome segments of HoVRV, and 21nt vsRNAs were most common. However, strikingly contrasting patterns for the HoCV-1 and HoVRV genomic RNAs were observed. The majority of HoCV-1 vsRNAs mapped to the genomic positive-strand RNA and, although minor hotspots were observed, vsRNAs mapped across the entire genomic RNA. In contrast, HoVRV vsRNAs mapped to both positive and negative-sense strands for all genome segments, but different genomic segments showed distinct hotspots. The HoVRV vsRNAs were more common for 5 and 3 regions of HoVRV regions of all segments. These data suggest that taxonomically different viruses in the same host offer different targets for RNA-antiviral defense.
Wheat streak mosaic virus (WSMV) is an eriophyid mite-transmitted virus of the genus Tritimovirus, family Potyviridae. Complete deletion of helper component-proteinase (HC-Pro) has no effect on WSMV virulence or disease synergism, suggesting that a different viral protein suppresses RNA silencing. RNA silencing suppression assays using Nicotiana benthamiana 16C plants expressing GFP were conducted with each WSMV protein; only P1 suppressed RNA silencing. Accumulation of GFP siRNAs was markedly reduced in leaves infiltrated with WSMV P1 at both 3 and 6 days post infiltration relative to WSMV HC-Pro and the empty vector control. On the other hand, helper component-proteinase (HC-Pro) of two species in the mite-transmitted genus Rymovirus, family Potyviridae was demonstrated to be a suppressor of RNA silencing. Symptom enhancement assays were conducted by inoculating Potato virus X (PVX) onto transgenic N. benthamiana. Symptoms produced by PVX were more severe on transgenic plants expressing WSMV P1 or potyvirus HC-Pro compared to transgenic plants expressing GFP or WSMV HC-Pro.
A complex set of double-stranded RNAs (dsRNAs) was isolated from threecornered alfalfa hopper (Spissistilus festinus), a plant-feeding hemipteran pest. A subset of these dsRNAs constitute the genome of a new reovirus, provisionally designated Spissistilus festinus reovirus (SpFRV). SpFRV was present in threecornered alfalfa hopper populations in the San Joaquin Valley of California, with incidence ranging from 10% to 60% in 24 of 25 sample sets analyzed. The 10 dsRNA segments of SpFRV were completely sequenced and shown to share conserved terminal sequences (5-AGAGA and CGAUGUUGU-3) of the positive-sense strand that are distinct from known species of the family Reoviridae. Comparisons of the RNA directed RNA polymerase (RdRp) indicated SpFRV is most closely related (39.1% amino acid identity) to another new reovirus infecting the angulate leafhopper (Acinopterus angulatus) and provisionally designated Acinopterus angulatus reovirus (AcARV). The RdRp of both viruses was distantly related to Raspberry latent virus RdRp at 27.0% (SpFRV) and 30.0% (AcARV) or Rice ragged stunt virus RdRp at 26.2% (SpFRV) and 29.0% (AcARV) amino acid identity. RdRp phylogeny confirmed that SpFRV and AcARV are sister taxa sharing a most recent common ancestor. SpFRV segment 6 encodes a protein containing two NTP binding motifs that are conserved in homologs of reoviruses in the subfamily Spinareovirinae. The protein encoded by SpFRV segment 4 was identified as a guanylyltransferase homolog. SpFRV segments 1, 3, and 10 encode homologs of reovirus structural proteins. No homologs were identified for proteins encoded by SpFRV segments 5, 7, 8, and 9. Collectively, the low level of sequence identity with other reoviruses, similar segment terminal sequences, RdRp phylogeny, and host taxa indicate that SpFRV and AcARV may be considered members of a proposed new genus of the family Reoviridae (subfamily Spinareovirinae), with SpFRV assigned as the type species.
Incompatibility group P1 (IncP-1) plasmid diversity was evaluated based on replication initiator protein (TrfA) phylogeny. A new and highly divergent clade was identified. Replication assays indicated that TrfA of recently discovered IncP-1 plasmids from Xylella fastidiosa and Verminephrobacter eiseniae initiated plasmid replication using cognate or heterologous origins of replication.
Xylella fastidiosa strain riv11 harbors a 25-kbp plasmid (pXF-RIV11) belonging to the IncP-1 incompatibility group. Replication and stability factors of pXF-RIV11 were identified and used to construct plasmids able to replicate in X. fastidiosa and Escherichia coli. Replication in X. fastidiosa required a 1.4-kbp region from pXF-RIV11 containing a replication initiation gene (trfA) and the adjacent origin of DNA replication (oriV). Constructs containing trfA and oriV from pVEIS01, a related IncP-1 plasmid of the earthworm symbiont Verminephrobacter eiseniae, also were competent for replication in X. fastidiosa. Constructs derived from pXF-RIV11 but not pVEIS01 replicated in Agrobacterium tumefaciens, Xanthomonas campestris, and Pseudomonas syringae. Although plasmids bearing replication elements from pXF-RIV11 or pVEIS01 could be maintained in X. fastidiosa under antibiotic selection, removal of selection resulted in plasmid extinction after 3 weekly passages. Addition of a toxin-antitoxin addiction system (pemI/pemK) from pXF-RIV11 improved plasmid stability such that >80 to 90% of X. fastidiosa cells retained plasmid after 5 weekly passages in the absence of antibiotic selection. Expression of PemK in E. coli was toxic for cell growth, but toxicity was nullified by coexpression of PemI antitoxin. Deletion of N-terminal sequences of PemK containing the conserved motif RGD abolished toxicity. In vitro assays revealed a direct interaction of PemI with PemK, suggesting that antitoxin activity of PemI is mediated by toxin sequestration. IncP-1 plasmid replication and stability factors were added to an E. coli cloning vector to constitute a stable 6.0-kbp shuttle vector (pXF20-PEMIK) suitable for use in X. fastidiosa.
Plant cytochrome P450 monooxygenases (CYP) mediate synthesis and metabolism of many physiologically important primary and secondary compounds that are related to plant defense against a range of pathogenic microbes and insects. To determine if cytochrome P450 monooxygenases are involved in defense response to Xylella fastidiosa (Xf) infection, we investigated expression and regulatory mechanisms of the cytochrome P450 monooxygenase CYP736B gene in both disease resistant and susceptible grapevines.
As RNA viruses evolve rapidly, we hypothesized that a virus could serve as a surrogate to discriminate recently separated populations of an invasive insect species. Homalodisca vitripennis reovirus (HoVRV) was used as a surrogate to assess population structure of glassy-winged sharpshooter (GWSS), an invasive species detected in California ~20 years ago. HoVRV nucleotide sequence polymorphism revealed a bottleneck in the introduced population, yielded population age estimates consistent with timing of GWSS discovery in California, suggested gene flow within the native range but not among native and introduced populations, and could potentially pinpoint source of the introduced population. Collectively, the data support use of a virus surrogate to define critical attributes of invasive species populations, with the caveat that life history of the surrogate must be closely linked to that of the host.
Novel double-stranded RNAs (approximately 8 kbp) were isolated from threecornered alfalfa hopper (Spissistilus festinus) and beet leafhopper (Circulifer tenellus), two plant-feeding hemipteran insect pests. The two new viruses, designated Spissistilus festinus virus 1 (SpFV1) and Circulifer tenellus virus 1 (CiTV1), do not appear to be encapsidated in conventional virions and shared a genome organization similar to that of several unclassified fungal viruses. SpFV1 and CiTVl encode a proline-alanine rich protein (PArp) and an RNA-directed RNA polymerase (RdRp). Expression of the 3-proximal RdRp ORF appears to result from -1 translational frameshifting of the PArp ORF. Phylogenetic analysis of the RdRp indicated that SpFV1 and CiTV1 were most closely related to each other and the unclassified plant virus Cucurbit yellows associated virus, and more distantly related to the unclassified fungal dsRNA viruses Phlebiopsis gigantea virus 2 and Fusarium graminearum virus 3.
A new virus species of the genus Phytoreovirus was isolated from glassy-winged sharpshooter (GWSS), Homalodisca vitripennis Germar (Hemiptera: Cicadellidae), in California and designated here as Homalodisca vitripennis reovirus (HoVRV). Extraction of nucleic acid from GWSS adults collected from three Californian populations revealed an array of double-stranded (ds) RNA species that was soluble in 2 M LiCl and resistant to degradation upon exposure to S1 nuclease and DNase. Analysis of nucleic acid samples from single GWSS adults indicated that HoVRV dsRNA accumulated to high titer in individual insects. Double-shelled isometric virus particles purified from GWSS adults resembled those observed in thin sections of GWSS salivary glands by transmission electron microscopy. Purified HoVRV virions contained 12 dsRNA segments that, based on complete nucleotide sequences, ranged in size from 4475 to 1040 bp. Sequence comparisons indicated that the HoVRV dsRNA segments were most closely related (58.5 to 43.7% nt sequence identity) to the corresponding genome segments of Rice dwarf virus (RDV). Each HoVRV dsRNA segment encoded a single open reading frame (>300 nts) except for segment 11, which appears to be dicistronic. Terminal nucleotide sequences of HoVRV positive-sense RNAs were similar to other phytoreoviruses (GGCG or GGCA at the 5-end and UGAU or CGAU at the 3-end) with adjacent imperfect inverted repeats potentially able to base pair. Phylogenetic analyses of the RNA-directed RNA polymerase (encoded by segment 1) and the outer capsid protein (encoded by segment 8) confirmed placement of HoVRV as a species of the genus Phytoreovirus sharing a most recent common ancestor with RDV. Reverse transcription-polymerase chain reaction assays revealed that HoVRV infection of GWSS in California was common and that the virus also occurred in GWSS populations from the Carolinas and Texas.
Wheat streak mosaic virus (WSMV) was first detected in Argentina in 2002. Comparison of 78 WSMV coat protein sequences revealed that three Argentine isolates were closely related to isolates from the American Pacific Northwest (APNW) and Australia. Complete sequences were determined for one Argentine isolate, four APNW isolates, and three additional isolates from other regions of the USA. Comparison of these eight new sequences with five previously sequenced isolates of WSMV confirmed close affinity of WSMV from Argentina with APNW isolates. Collectively, these results indicate concurrent establishment of the same WSMV lineage in both Argentina and Australia.
A ? 38kB plasmid (pXF-RIV5) was present in the Riv5 strain of Xylella fastidiosa subsp. multiplex isolated from ornamental plum in southern California. The complete nucleotide sequence of pXF-RIV5 is almost identical to that of pXFAS01 from X. fastidiosa subsp. fastidiosa strain M23; the two plasmids vary at only 6 nucleotide positions. BLAST searches and phylogenetic analyses indicate pXF-RIV5 and pXFAS01 share some similarity to chromosomal and plasmid (pXF51) sequences of X. fastidiosa subsp. pauca strain 9a5c and more distant similarity to plasmids from a wide variety of bacteria. Both pXF-RIV5 and pXFAS01 encode homologues of a complete Type IV secretion system involved in conjugation and DNA transfer among bacteria. Mating pair formation proteins (Trb) from Yersinia pseudotuberculosis IP31758 are the mostly closely related non-X. fastidiosa proteins to most of the Trb proteins encoded by pXF-RIV5 and pXFAS01. Unlike many bacterial conjugative plasmids, pXF-RIV5 and pXFAS01 do not carry homologues of known accessory modules that confer selective advantage on host bacteria. However, both plasmids encode seven hypothetical proteins of unknown function and possess a small transposon-associated region encoding a putative transposase and associated factor. Vegetative replication of pXF-RIV5 and pXFAS01 appears to be under control of RepA protein and both plasmids have an origin of DNA replication (oriV) similar to that of pRP4 and pR751 from Escherichia coli. In contrast, conjugative plasmids commonly encode TrfA and have an oriV similar to those found in IncP-1 incompatibility group plasmids. The presence of nearly identical plasmids in single strains from two distinct subspecies of X. fastidiosa is indicative of recent horizontal transfer, probably subsequent to the introduction of subspecies fastidiosa to the United States in the late 19(th) century.
Xylella fastidiosa, the causal agent of Pierces disease of grapevine, possesses several two-component signal transduction systems that allow the bacterium to sense and respond to changes in its environment. Signals are perceived by sensor kinases that autophosphorylate and transfer the phosphate to response regulators (RRs), which direct an output response, usually by acting as transcriptional regulators. In the X. fastidiosa genome, 19 RRs were found. A site-directed knockout mutant in one unusual RR, designated XhpT, composed of a receiver domain and a histidine phosphotransferase output domain, was constructed. The resulting mutant strain was analysed for changes in phenotypic traits related to biofilm formation and gene expression using microarray analysis. We found that the xhpT mutant was altered in surface attachment, cell-cell aggregation, exopolysaccharide (EPS) production and virulence in grapevine. In addition, this mutant had an altered transcriptional profile when compared with wild-type X. fastidiosa in genes for several biofilm-related traits, such as EPS production and haemagglutinin adhesins.
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