Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 211 - Insects, Mites, and Other Arthropods Affecting Plants | 3110 - Insects | 1040 - Molecular biology | 60% |
| 212 - Pathogens and Nematodes Affecting Plants | 4030 - Viruses | 1101 - Virology | 40% |
Many insects feed on plants of agricultural and/or environmental importance. Some insects also serve as vectors to spread specific plant pathogens, including plant viruses, from plant to plant. Due to losses directly due to insect pests, and indirectly from their propensity to spread plant pathogens, there is a great need for more effective and environmentally sound strategies to complement existing approaches used for controlling these insects, and for the pathogens they transmit to plants. This project will evaluate and then attempt to utilize new approaches, including RNA interference (RNAi) to induce negative effects on plant feeding insects. RNAi approaches will also be evaluated against specific plant-infecting viruses. We believe that we can use RNAi as a tool to target and assist in the control insectpests and plant virus vectors in an environmentally sound and sustainable manner. We will investigate new opportunities by performing large scale sequencing and bioinformatics to identify candidate RNA targets in insects. Bioassays and molecular biological analyses will be used to assess effectiveness of interfering RNAs (and in some cases proteins). In the long term we envision developing transgenic plants engineered to express specific interfering molecules that can confer insect vector and/or virus resistance. We believe that this offers a potentially long-term, environmentally sound strategy for helping to control plant feeding hemipterans and the pathogens they transmit to plants.
Our primary plant-feeding hemipterans of study include Bemisia tabaci (sweet potato whitefly); Homalodisca vitripennis (glassy-winged sharpshooter); Bactericera cockerelli (potato Psyllid); and Diaphorina citri (Asian citrus Psyllid). B. tabaci transmits several very important plant viruses, and of these we are studying Lettuce infectious yellows virus (LIYV); Cucurbit yellow stunting disorder virus (CYSDV), Sweet potato chlorotic stunt virus (SPSCV), Cucurbit chlorotic yellows virus (CCYV), and Tomato apex necrosis virus (ToANV). Anti-plant feeding hemipteran strategies: We will use next generation Illumina-based sequencing strategies to identify hemipteran sequences that may serve as targets for anti-insect RNA and/or protein based approaches. In the case of D. citri, we will collect and sequence worldwide populations, therefore we will also obtain representative microbe (e.g. virus) sequences from the different D. citri populations. Insect sequences will be assembled into transcriptome datasets. Sequences will be compared with those of other insects and used to identify targets to evaluate by RNAi-based approaches. This includes identifying optimal RNA candidates, but also ensuring that the target and inducer RNAi sequences are specific to the target insect thereby minimizing opportunities for potential off-target effects. We will assess efficacy of candidate interfering RNA and protein sequences by in vitro and in planta approaches. We will use artificial-diet-based approaches to delivery candidates for in vitro studies. This is straightforward, we have done it previously. Potential effects will be assessed by measuring target insect mortality but for RNAi approaches also assessing target mRNA knockdown relative to non-target RNA standards. This will be done by RT-qPCR and by small RNA northern hybridization analyses. In planta experiments will be done using recombinant plant viruses initially as the means to express candidate RNAs and proteins in plants. Efficacy will be measured by mortality, fecundity and in the case of RNAi, by using RT-qPCR as above. Candidates that show good potential in these assays will be used to generate transgenic plants. These will then be used as above.Because Bemisia tabaci is also a vector of several important plant-infecting viruses, we also will target the five viruses above. So far no RNAi-based immunity has been reported for any crinivirus (this includes LIYV, CYSDV, SPSCV and CCYV). Our preliminary studies have shown that RNAi-based approaches to target specific LIYV RNA 1 sequences can yield RNAi effects, including immunity, in transgenic plants. Therefore, we will assess if similar strategies prove effective against the other criniviruses here: SPSCV, CCYV and CYSDV. We will generate transgenic squash and sweet potato plants (depending on the virus) and evaluate resistance after plants are challenged by B. tabaci-mediated inoculation. Resistance will be scored by phenotypic responses, but also using northern blot hybridization analysis for virus-specific large and small RNAs.There are currently no reports in the literature regarding resistance to torradoviruses such as ToANV. ToANV is a member of the genus Torradovirus; these whitefly-transmitted plant viruses are emerging newly described viruses and various viruses affect a number of different, important food crops. Like criniviruses, Torradoviruses have bipartite RNA genomes. We will generate transgenic plants with specific constructs corresponding to different regions of each of the genomic RNAs. Transgenic plants will be assessed for resistance and susceptibility by whitefly-mediated virus inoculations. Resistance and susceptibility will be scored as above. Based on the results from our combined approaches here, if we find successes for B. tabaci and criniviruses, we will attempt to develop plants that are resistant to B. tabaci and the criniviruses it transmits to squash (CYSDV and CCYV).
Target Audience
The target audiences for the work here include California and Florida citrus growers, entomologists, plant pathologists, plant virologists and agricultural scientists in the U.S. and around the world. We presented results at annual American Society for Virology meetings each year during this project. We also presented results at the International Organization of Citrus Virologists meeting in 2019, and in seminars in several university academic departments.
Changes / Problems
Nothing Reported
Training & Professional Development
I had 7 postdoctoral scientists and 7 graduate students in my lab during this project. Each worked on various aspects of this project, all gained new knowledge, generated scientific publications and got to work as a team. In addition I had approximately 4 UC Davis undergraduate students each year who helped work on specific aspects of our research.
Dissemination Streams
We disseminated results via the publications given above and in previous annual reports. We also presented abstracts and oral talks at the meetings and seminars as mentioned above. Quarterly and annual reports also have been submitted to the appropriate funding agencies.
Next Reporting Steps
Nothing Reported
Target Audience
The target audiences for the work here include California and Florida citrus growers, entomologists, plant pathologists, plant virologists and agricultural scientists in the U.S. and around the world. We presented results at the American Society for Virology annual meeting in College Park, MD, July 2018.
Changes / Problems
Nothing Reported
Training & Professional Development
I had 4 postdoctoral scientists and 5 graduate students in my lab during the past year, who worked on various aspects of this project. In addition 4 UC Davis undergraduates worked on specific aspects of our research.
Dissemination Streams
We disseminated results via the publications given above. We also presented abstracts and oral talks at the meeting mentioned above. Quarterly reports also have been submitted to the appropriate funding agencies.
Next Reporting Steps
We will continue our efforts along the same lines. We hope to begin more translational applications of our research. <br><br>
<br>What was accomplished under these goals? We accomplished identification of RNAs to target the Asian citrus psyllid, Diaphorina citri, we developed a phloem-limited virus as a vehicle to express RNAs and proteins in plants, we reviewed the opportunities of using RNA interference (RNAi) for plant pathogen and insect studies and we presented new ideas on using insect-specific viruses for beneficial targreting of insect vectors of plant pathogens. Plant virus-based vectors are valuable tools for recombinant gene expression and functional genomics for both basic and applied research. In this study, Lettuce infectious yellows virus (LIYV) of the genus Crinivirus 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 Nicotiana benthamiana 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, phytoene desaturase (PDS) in N. benthamiana plants, and the transgene GFP in N. benthamiana line 16c plants. Therefore, LIYV-derived vectors could provide a technical reference for developing vectors of other economically important criniviruses. The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is one of the most important citrus pests. It is the vector of the phloem-limited bacteria Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus, the causal agents of the devastating citrus disease, huanglongbing (HLB). The management of HLB is based on the use of healthy young plants, eradication of infected plants and chemical control of the vector. RNA interference (RNAi) has proven to be a promising tool to control pests as well as to explore gene functions. Recently, it has been reported that gene knockdown in many insects can be induced through feeding with dsRNA. In the current study, we targeted cathepsin D, chitin synthase and inhibitor of apoptosis genes of adult and nymph ACP by artificial diets and Murraya paniculata leaves, respectively. Adult ACP mortality was positively correlated with the amount of dsRNA used. Both nymphs and adult ACP fed dsRNAs exhibited significantly increased mortality over time compared to controls. Moreover, qRT-PCR analysis confirmed the dsRNA-mediated RNAi effects on target mRNAs. These results showed that RNAi can be a powerful tool for gene function studies in ACP and perhaps HLB control. Lettuce infectious yellows virus (LIYV), genus Crinivirus, family Closteroviridae, has long, filamentous flexuous virions, and causes phloem-limited infections in its plant hosts. The LIYV-encoded P26 is a distinct non-virion protein which shows no similarities to proteins in current databases, it induces plasmalemma deposits over plasmadesmata 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 in planta by mutagenesis analysis. An LIYV P26 knockout mutant (P26X) showed viral RNA replication and virion formation in inoculated leaves of Nicotiana benthamiana plants, but failed to give systemic infection. Confirmation by using a modified GFP-tagged LIYV P26X showed GFP accumulation only in infiltrated leaf tissues while wildtype LIYV GFP readily spread systemically in the phloem. Attempts to rescue P26X by complementation in trans were negative. However a translocated LIYV P26 gene in the LIYV genome rescued systemic infection, but P26 orthologs from other criniviruses did not. Mutagenesis in planta assays showed that deletions in P26, as well as two of 11 specific alanine-scanning mutants, abolished the ability to systemically infect N. benthamiana. Over the past decade the scientific community have experienced a new age of virus discovery in arthropods in general, and in insects in particular. Next generation sequencing and advanced bioinformatics tools have provided new insights about insect viromes and viral evolution. Additionally, the discovery of these novel viruses that are considered as insect-specific viruses has gained increasing attention in their potential use as biological agents. We focussed on high-throughput sequencing technologies used to discover viruses in insects and the challenges raised in data interpretations. We also discussed some potential translational applications of insect-specific viruses, both natural or engineered, to limit vector competence of important insect vectors or target pathogens transmitted by them. <br><br><b>Publications</b><br>
Target Audience
The target audiences for the work here include California and Florida citrus growers, entomologists, plant pathologists, plantvirologists and agricultural scientists in the U.S. and around the world. We also presented results at the American Societyfor Virology annual meeting in Madison WI, and at the International Citrus meeting in Brazil.
Changes / Problems
N/A
Training & Professional Development
The graduate student and 2 of the postdocs on this project were able to attend and participate in the Annual Meeting of the American Society for Virology in Madison, WI. In addition, an undergraduate student was able to participate in aspects of this project and learn what it is like to work in a science lab.
Dissemination Streams
We disseminated results via publication in scientific journals as noted above, and via presentations at the scientific meetings listed.
Next Reporting Steps
We will continue our efforts and make further progress towards achieving our goals. <br><br>
<br>What was accomplished under these goals? We provided direct evidence for the semipersistent transmission of Cucumber chlorotic yellows virus (CCYV) by B. tabaci Mediterranean cryptic species (MED, known as Q biotype). We investigated CCYV transmission characteristics, and immunofluorescently labeled and localized the virus retention site within the whitefly vector by laser confocal microscopy. Whitefly vectors required ≥1 h of acquisition access period (AAP) to successfully acquire CCYV, and the proportion of RT-PCR positive whitefly individuals reached to 100% at 48 h of AAP. CCYV virons could be retained within B. tabaci as long as 12 d, but the proportion of RT-PCR positive whiteflies dropped to 55% by 3 d. Groups of thirty whiteflies given a 24 h of inoculation access period (IAP) to inoculate CCYV on cucumber plants showed a transmission efficiency rate of 72.73%. The retention site of CCYV virons was located in the foregut of virion-fed vectors. These results definitely indicated the semipersistent transmission mode of CCYV by Bemisia tabaci Q biotype. We searched for viruses associated with Citrus sudden death (CSD). We performed a comparative high-throughput sequencing analysis of the transcriptome and small RNAs from CSD-symptomatic and -asymptomatic plants using the Illumina platform. The data revealed mixed infections that included Citrus tristeza virus (CTV) as the most predominant virus, followed by the Citrus sudden death-associated virus (CSDaV), Citrus endogenous pararetrovirus (CitPRV) and two putative novel viruses tentatively named Citrus jingmen-like virus (CJLV), and Citrus virga-like virus (CVLV). The deep sequencing analyses were sensitive enough to differentiate two genotypes of both viruses previously associated with CSD-affected plants: CTV and CSDaV. Our data also showed a putative association of the CSD-symptomatic plants with a specific CSDaV genotype and a likely association with CitPRV as well, whereas the two putative novel viruses showed to be more associated with CSD-asymptomatic plants. This is the first high-throughput sequencing-based study of the viral sequences present in CSD-affected citrus plants, and generated valuable information for further CSD studies. Tomato black ring virus (TBRV, genus Nepovirus) causes severe diseases in plants that are economically important such as tomato, potato, tobacco and cucumber. We successfully created the first infectious full-length cDNA clones of the TBRV genomic RNAs (RNA1 and RNA2). The engineered constructs consisting of PCR-amplified DNAs were cloned into binary vector pJL89 immediately downstream of a double Cauliflower mosaic virus (CaMV) 35S promoter, and upstream of the Hepatitis delta virus (HDV) ribozyme and nopaline synthase terminator (NOS). After agrobacterium-mediated co-inoculation of these two constructs, host plants developed a systemic infection and caused symptoms indistinguishable from wild-type infection. The presence of viral particles and TBRV RNA was confirmed by electron microscopy and reinoculation to Nicotiana tabacum var. Xanthi, Chenopodium quinoa and Cucumis sativus, as well as by reverse transcription polymerase chain reaction. The construction of full-length infectious cDNA clones will be an appealing way to investigate determinants of TBRV variability and study virus-host-vector interactions. <br><br><b>Publications</b><br>
Target Audience
The target audiences for the work here include California and Florida citrus growers, entomologists, plant pathologists, plant virologists and agricultural scientists in the U.S. and around the world. We also presented results at the American Society for Virology annual meeting in Blacksburg, VA in June 2016.
Changes / Problems
Nothing Reported
Training & Professional Development
I had 4 postdoctoral scientists, 4 graduate students and 2 visiting graduate students in my lab during the past year, who worked on various aspects of this project. In addition 3 UC Davis undergraduates worked on specific aspects of our research.
Dissemination Streams
We disseminated results via the publications given above. We also presented abstracts and oral talks at the meeting mentioned above. Quarterly reports also have been submitted to the appropriate funding agencies.
Next Reporting Steps
We will continue our efforts along the same lines. We hope to begin more translational applications of our research. <br><br>
<br>What was accomplished under these goals? We took efforts to better understand the Glassy-winged sharpshooter, Homalodisca vitripennis, the xylem-feeding vector of the bacterium Xylella fastidiosa. The glassy-winged sharpshooter (GWSS) Homalodisca vitripennis (Hemiptera: Cicadellidae), is a xylem-feeding leafhopper and an important vector of the bacterium Xylella fastidiosa; the causal agent of Pierce's disease of grapevines. MicroRNAs are a class of small RNAs that play an important role in the functional development of various organisms including insects. In H. vitripennis, we identified microRNAs using high-throughput deep sequencing of adults followed by computational and manual annotation. A total of 14 novel microRNAs that are not found in the miRBase were identified from adult H. vitripennis. Conserved microRNAs were also found in our datasets. By comparison to our previously determined transcriptome sequence of H. vitripennis, we identified the potential targets of the microRNAs in the transcriptome. This microRNA profile information not only provides a more nuanced understanding of the biological and physiological mechanisms that govern gene expression in H. vitripennis, but may also lead to the identification of novel mechanisms for biorationally designed management strategies through the use of microRNAs. We also assessed the population structure of Homalodisca coagulata Virus-1 (HoCV-1) among and within field-collected H. vitripennis sampled from a single point in space and time. The objective was to assess HoCV-1 variability as a means to understand how HoCV-1 (and other viruses) vary or are similar in their insect hosts. The population as a whole showed little variation. We found as much variation in a single insect as we did among different insects, suggesting that HoCV-1 is relatively stable. This information is important for our future efforts to use insect viruses as one means to modify biological activities of insect vectors of plant pathogens. <br><br><b>Publications</b><br>
Target Audience
The target audiences for the work here include California and Florida citrus growers, entomologists, plant pathologists, plant virologists and agricultural scientists in the U.S. and around the world. We also presented results at the American Society for Virology annual meeting in London, Ontario, Canada, and the American Phytopathological Society annual meeting in Pasadena, CA.
Changes / Problems
Nothing Reported
Training & Professional Development
I had 5 postdoctoral scientists, 4 graduate students and 3 visiting graduate students in my lab during the past year, who worked on various aspects of this project. In addition 3 UC Davis undergraduates worked on specific aspects of our research.
Dissemination Streams
We disseminated results via the publications given above. We also presented abstracts and oral talks at the meetings mentioned above. Quarterly reports also have been submitted to the appropriate funding agencies.
Next Reporting Steps
We will continue our efforts along the same lines. We hope to begin more translational applications of our research. <br><br>
<br>What was accomplished under these goals? Tomato torrado virus (ToTV) causes serious damage to tomato industry and significant economic losses. ToTV is whitefly transmitted to tomato, pepper, eggplants and tomatillo, and our efforts to understand how it interacts with its whitefly vector and plant hosts is ongoing. To that end, we developed a quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) method using primers and a specific TaqMan® 41 MGB probe for ToTV detection in plants. This method was developed for sensitive detection and quantitation of different ToTV isolates, with the intent to also apply it for viruliferous whitefly vectors. A standard curve using RNA transcripts enabled absolute quantitation, with a dynamic range from 104 -1010 ToTV RNA copies / ng of total RNA. RT-qPCR was assayed with twenty-three ToTV isolates from tomato (Solanum lycopersicum), and black nightshade (Solanum nigrum) collected in Spain, Australia, Hungary and France,which covered the genetic variation range of this virus. This new RT-qPCR assay enables a reproducible, sensitive and specific detection and quantitation of ToTV, which can be a valuable tool in disease management programs and epidemiological studies. We also focused efforts on the Asian citrus psyllid, Diaphorina citri. D. 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. 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 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. <br><br><b>Publications</b><br>