The citrus mealybug, Planococcus citri, is an important plant pest with a very broad plant host range. P. citri is a phloem feeder and loss of plant vigor and stunting are characteristic symptoms ...induced on a range of host plants, but P. citri also reduces fruit quality and causes fruit drop leading to significant yield reductions. Better strategies for managing this pest are greatly needed. RNA interference (RNAi) is an emerging tool for functional genomics studies and is being investigated as a practical tool for highly targeted insect control. Here we investigated whether RNAi effects can be induced in P. citri and whether candidate mRNAs could be identified as possible targets for RNAi-based P. citri control. RNAi effects were induced in P. citri, as demonstrated by specific target reductions of P. citri actin, chitin synthase 1 and V-ATPase mRNAs after injection of the corresponding specific double-stranded RNA inducers. We also used recombinant Tobacco mosaic virus (TMV) to express these RNAi effectors in Nicotiana benthamiana plants. We found that P. citri showed lower fecundity and pronounced death of crawlers after feeding on recombinant TMV-infected plants. Taken together, our data show that actin, chitin synthase 1 and V-ATPase mRNAs are potential targets for RNAi against P. citri, and that recombinant TMV is an effective tool for evaluating candidate RNAi effectors in plants.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Since its discovery in 2016, the Polerovirus Barley virus G has been reported in at least nine countries and multiple species of monocot plants. All of these reports have used PCR and/or sequencing ...based assays to identify BVG, however none have investigated the biology of BVG. In this study we detail the generation of the first infectious cDNA clone of BVG from archived RNA, thereby producing a valuable experimental tool and system for studying BVG biology. Using this system we identified two compatible aphid vectors and confirmed the susceptibility of several monocot plants, and the dicotyledonous plant host Nicotiana benthamiana, to BVG.
•Construction of an infectious cDNA clone of the emergent Polerovirus Barley virus G.•Rhopalosiphom maidis and R. padi aphids are vectors of BVG.•R. maidis is a highly efficient BVG vector.•Multiple monocot crop plants are susceptible to BVG infection.
Most plant viruses are absolutely dependent on a vector for plant-to-plant spread. Although a number of different types of organisms are vectors for different plant viruses, phloem-feeding ...Hemipterans are the most common and transmit the great majority of plant viruses. The complex and specific interactions between Hemipteran vectors and the viruses they transmit have been studied intensely, and two general strategies, the capsid and helper strategies, are recognized. Both strategies are found for plant viruses that are transmitted by aphids in a nonpersistent manner. Evidence suggests that these strategies are found also for viruses transmitted in a semipersistent manner. Recent applications of molecular and cell biology techniques have helped to elucidate the mechanisms underlying the vector transmission of several plant viruses. This review examines the fundamental contributions and recent developments in this area.
Plant viruses are emerging and re-emerging to cause important diseases in many plants that humans grow for food and/or fiber, and sustainable, effective strategies for controlling many plant virus ...diseases remain unavailable ....
The Asian citrus psyllid, Diaphorina citri, is the natural vector of the causal agent of Huanglongbing (HLB), or citrus greening disease. Together; HLB and D. citri represent a major threat to world ...citrus production. As there is no cure for HLB, insect vector management is considered one strategy to help control the disease, and D. citri viruses might be useful. In this study, we used a metagenomic approach to analyze viral sequences associated with the global population of D. citri. By sequencing small RNAs and the transcriptome coupled with bioinformatics analysis, we showed that the virus-like sequences of D. citri are diverse. We identified novel viral sequences belonging to the picornavirus superfamily, the Reoviridae, Parvoviridae, and Bunyaviridae families, and an unclassified positive-sense single-stranded RNA virus. Moreover, a Wolbachia prophage-related sequence was identified. This is the first comprehensive survey to assess the viral community from worldwide populations of an agricultural insect pest. Our results provide valuable information on new putative viruses, some of which may have the potential to be used as biocontrol agents.
Insects have the most species of all animals, and are hosts to, and vectors of, a great variety of known and unknown viruses. Some of these most likely have the potential to be important fundamental and/or practical resources. In this study, we used high-throughput next-generation sequencing (NGS) technology and bioinformatics analysis to identify putative viruses associated with Diaphorina citri, the Asian citrus psyllid. D. citri is the vector of the bacterium causing Huanglongbing (HLB), currently the most serious threat to citrus worldwide. Here, we report several novel viral sequences associated with D. citri.
Small RNAs serve as specificity determinants of antiviral responses in insects. Piwi-interacting RNAs (piRNAs) are a class of small RNAs found in animals, and their primary role is to direct ...antitransposon responses. These responses require endogenous piRNAs complementary to transposon RNA. Additionally, piRNAs have been shown to target RNA and DNA viruses in some mosquito species. In contrast to transposons, targeting of viruses by the piRNA pathway in these mosquito species does not require endogenous piRNAs. Here, we show that piRNAs target a DNA virus, but not RNA viruses, in an agricultural insect pest. We found that targeting of this DNA virus did not require endogenous piRNAs and that endogenous piRNAs did not mediate targeting of an RNA virus with which they shared complementary sequence. Our results highlight differences between mosquitoes and our experimental system and raise the possibility that DNA viruses may be targeted by piRNAs in other species.
ABSTRACT
Piwi-interacting RNAs (piRNAs) are a class of small RNAs primarily responsible for silencing transposons in the animal germ line. The ping-pong cycle, the posttranscriptional silencing branch of the piRNA pathway, relies on piRNAs produced from endogenous transposon remnants to direct cleavage of transposon RNA via association with Piwi-family Argonaute proteins. In some mosquito species and mosquito-derived cell lines expressing a functionally expanded group of Piwi-family Argonaute proteins, both RNA and DNA viruses are targeted by piRNAs in a manner thought to involve direct processing of exogenous viral RNA into piRNAs. Whether viruses are targeted by piRNAs in nonmosquito species is unknown. Partial integrations of DNA and nonretroviral RNA virus genomes, termed endogenous viral elements (EVEs), are abundant in arthropod genomes and often produce piRNAs that are speculated to target cognate viruses through the ping-pong cycle. Here, we describe a Diaphorina citri densovirus (DcDV)-derived EVE in the genome of
Diaphorina citri
. We found that this EVE gives rise to DcDV-specific primary piRNAs and is unevenly distributed among
D. citri
populations. Unexpectedly, we found that DcDV is targeted by ping-pong-dependent virus-derived piRNAs (vpiRNAs) in
D. citri
lacking the DcDV-derived EVE, while four naturally infecting RNA viruses of
D. citri
are not targeted by vpiRNAs. Furthermore, a recombinant Cricket paralysis virus containing a portion of the DcDV genome corresponding to the DcDV-derived EVE was not targeted by vpiRNAs during infection in
D. citri
harboring the EVE. These results demonstrate that viruses can be targeted by piRNAs in a nonmosquito species independently of endogenous piRNAs.
IMPORTANCE
Small RNAs serve as specificity determinants of antiviral responses in insects. Piwi-interacting RNAs (piRNAs) are a class of small RNAs found in animals, and their primary role is to direct antitransposon responses. These responses require endogenous piRNAs complementary to transposon RNA. Additionally, piRNAs have been shown to target RNA and DNA viruses in some mosquito species. In contrast to transposons, targeting of viruses by the piRNA pathway in these mosquito species does not require endogenous piRNAs. Here, we show that piRNAs target a DNA virus, but not RNA viruses, in an agricultural insect pest. We found that targeting of this DNA virus did not require endogenous piRNAs and that endogenous piRNAs did not mediate targeting of an RNA virus with which they shared complementary sequence. Our results highlight differences between mosquitoes and our experimental system and raise the possibility that DNA viruses may be targeted by piRNAs in other species.
The Asian citrus psyllid, Diaphorina citri Kuwayama, is an important insect vector of Candidatus Liberibacter asiaticus, the causal agent of Huanglongbing, which is the most destructive disease of ...citrus worldwide. Sequences for putative Diaphorina citri reovirus (DcRV) were identified from some worldwide populations of D. citri. Here, field surveys indicated that the virus was common in D. citri populations from Hawaii and Fuzhou of PR China. Electron microscopy showed that DcRV virions possessed a typical reovirus-like morphology. The U. S. and Chinese DcRV isolates both showed 10 segments of double-stranded RNA sharing >96% nucleotide sequence identity, and encoding 11 deduced proteins. All genome segments contained conserved 5′ and 3′ terminal nucleotide sequences and inverted repeats that are hallmarks of reovirus sequence. Phylogenetic analysis showed that DcRV may be considered a new species of the genus Fijivirus sharing a most recent common ancestor with the insect-specific fijivirus Nilaparvata lugens reovirus.
Diaphorina citri densovirus (DcDV) is an ambisense densovirus with a 5071 nt genome. Phylogenetic analysis places DcDV in an intermediate position between those in the
and
genera, a finding that is ...consistent with the observation that DcDV possesses an
-like non-structural (NS) protein-gene cassette, but a capsid-protein (VP) gene cassette resembling those of other ambisense densoviruses. DcDV is maternally transmitted to 100 % of the progeny of infected female
, and the progeny of infected females carry DcDV as a persistent infection without outward phenotypic effects. We were unable to infect naïve individuals by oral inoculation, however low levels of transient viral replication are detected following intrathoracic injection of DcDV virions into uninfected
insects. Transcript mapping indicates that DcDV produces one transcript each from the NS and VP gene cassettes and that these transcripts are polyadenylated at internal sites to produce a ~2.2 kb transcript encoding the NS proteins and a ~2.4 kb transcript encoding the VP proteins. Additionally, we found that transcriptional readthrough leads to the production of longer non-canonical transcripts from both genomic strands.
Plant-infecting viruses utilize various strategies involving multiple viral and host factors to achieve successful systemic infections of their compatible hosts.
(LIYV), genus
, family
, has long, ...filamentous flexuous virions and causes phloem-limited infections in its plant hosts. The LIYV-encoded P26 is a distinct non-virion protein that shows no similarities to proteins in current databases: it induces plasmalemma deposits over plasmadesmata (PD) pit fields and is speculated to have roles in LIYV virion transport within infected plants. In this study, P26 was demonstrated to be a PD-localized protein, and its biological significance was tested
by mutagenesis analysis. An LIYV P26 knockout mutant (P26X) showed viral RNA replication and virion formation in inoculated leaves of
plants, but failed to give systemic infection. Confirmation by using a modified green fluorescent protein (GFP)-tagged LIYV P26X showed GFP accumulation only in infiltrated leaf tissues, while wild-type LIYV GFP readily spread systemically in the phloem. Attempts to rescue P26X by complementation in
were negative. However a translocated LIYV P26 gene in the LIYV genome rescued systemic infection, but P26 orthologs from other criniviruses did not. Mutagenesis
assays showed that deletions in P26, as well as 2 of 11 specific alanine-scanning mutants, abolished the ability to systemically infect
Plant viruses encode specific proteins that facilitate their ability to establish multicellular/systemic infections in their host plants. Relatively little is known of the transport mechanisms for plant viruses whose infections are phloem limited, including those of the family
These viruses have complex, long filamentous virions that spread through the phloem.
(LIYV) encodes a non-virion protein, P26, which forms plasmalemma deposits over plasmodesmata pit fields, and LIYV virions are consistently found attached to those deposits. Here we demonstrate that P26 is a unique movement protein required for LIYV systemic infection in plants. LIYV P26 shows no sequence similarities to other proteins, but other criniviruses encode P26 orthologs. However, these failed to complement movement of LIYV P26 mutants.
Plant virus-based vectors are valuable tools for recombinant gene expression and functional genomics for both basic and applied research. In this study,
(LIYV) of the genus
was engineered into a ...virus vector that is applicable for efficient protein expression and virus-induced gene silencing (VIGS) in plants. We examined gene replacement and "add a gene" strategies to develop LIYV-derived vectors for transient expression of the green fluorescent protein (GFP) reporter in
plants. The latter yielded higher GFP expression and was further examined by testing the effects of heterologous controller elements (CEs). A series of five vector constructs with progressively extended LIYV CP sgRNA CEs were tested, the longest CE gave the highest GFP expression but lower virus accumulation. The whitefly transmissibility of the optimized vector construct to other host plants, and the capability to accommodate and express a larger gene, a 1.8 kb β-glucuronidase (GUS) gene, were confirmed. Furthermore, the LIYV vector was also validated VIGS by silencing the endogenous gene,
(PDS) in
plants, and the transgene GFP in
line 16c plants. Therefore, LIYV-derived vectors could provide a technical reference for developing vectors of other economically important criniviruses.
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