Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Plant Pathology
Non Technical Summary
This project supports the mission of the Agricultural Experiment Station by addressing the Hatch Act areas of plant and animal production, protection and health; sustainable agriculture, molecular and biotechnology. Viruses are the most abundant microbes on our planet and our project will attempt to use plant and insect viruses as beneficial tools to help manage plant diseases by target insect vectors of plant pathogens. We will evaluate and utilize new approaches, including RNA interference (RNAi) to induce desired effects on plant feeding insects. We believe that we can use RNAi as a tool to target and assist in the control insect pests and plant virus vectors in an environmentally sound and sustainable manner. Bioassays and molecular biological analyses will be used to assess effectiveness of interfering RNAs (and in some cases proteins). We believe that this project offers a potentially long-term, environmentally sound strategy for helping to control plant feeding hemipterans and the pathogens they transmit to plants.
Animal Health Component
20%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Goals / Objectives
Our long-term goals are to gain new understanding of fundamental processes affecting plant pests and pathogens, and to ultimately be in position to use the information for developing new, environmentally sound, sustainable approaches for protecting plants. Our previous work has been directed mostly towards plant viruses. Here we continue efforts with plant viruses but also target the insect vectors of some plant viruses and of some plant pathogenic prokaryotes. We will use plant and insect viruses as delivery tools to target insect vectors of plant pathogens. Our lab has extensive experience with aphids, whiteflies, leafhoppers, planthoppers, thrips psyllids and even treehoppers as vectors of plant viruses and prokaryotic plant pathogens. Thus, we are uniquely positioned to take advantage of the opportunities presented in this proposal.The specific objectives of our research proposed here are:1) To develop and evaluate candidate plant viruses as vehicles for delivering proteins and RNAs in plants to target hemipteran vectors of plant pathogens.2) To identify optimal forms of interfering RNAs to be used in transgenic plants for targeting hemipteran vectors of plant pathogens.3) To evaluate using insect viruses to deliver interfering RNAs and proteins to target hemipteran vectors of plant pathogens.
Project Methods
Objective 1. We will use infectious clones of common plant viruses, Tobacco mosaic virus (TMV), Tomato mottle virus (ToMoV), Citrus tristeza virus (CTV) and Citrus sudden death-associated virus (CSDaV) and engineer them to express desired sequences in plants. Our target insects include the Asian citrus psyllid, Diaphorina citri, and various aphid vectors of plant viruses. We will next perform insect RNAi assays as we have done previously (Rosa, Kamita et al. 2012, Khan, Ashfaq et al. 2013, Wuriyanghan and Falk 2013), and examine insects for RNAi-based biological (e.g. mortality, reduced fecundity) and target mRNA knockdown effects. If successful, recombinant viruses offer the opportunity for a continuous supply of small interfering RNAs in non-transgenic plants.Objective 2. Most transgenic plant-based RNAi strategies are based on producing a hairpin dsRNA in the transgenic plant. However, when transgenic plants are engineered to produce dsRNAs targeting an insect mRNA, this is not the natural plant defense towards the insect. Furthermore, it is very likely that the transgenic plant-produced dsRNA is not the only transgene-derived RNAi inducer produced. The transgenically-generated dsRNAs are produced, but they are recognized by the plant's existing RNAi defenses (AGO1, DCL1, DCL3, DCL4), and then just like any other dsRNA, they are processed into siRNAs (Melnyk, Molnar et al. 2011). Because of the mechanism for RNAi-based dsRNA degradation in the plant cytoplasm, the transgenically-generated dsRNAs are processed to yield a complex population of overlapping siRNAs, all of which are homologous to the target insect mRNA, but some may have greater or lesser homology to other insect mRNAs. Furthermore, an important point is that RNAi-based processing of transgene-derived RNAs in plants does not occur at distinct, phased ~21 nt intervals. Rather the transgene-derived dsRNA sequence can be cleaved by Dicer beginning at essentially any nucleotide, yielding a population of overlapping siRNAs ranging in size (ca. 21 - 24 most commonly) and sequence corresponding to the target sequence (Ding and Lu 2011). Then an important question is: Do these resulting siRNAs increase the probability of risk due to potential off-target effects among other insects? One of the criticisms of RNAi-based approaches for insect control in plants is that we need to ensure that we eliminate the potential for unwanted off-target RNAi effects that may occur insects other than the pests.One way to do this is to first identify and then only express the minimal interfering RNA in plants. Here we will evaluate methods for expressing only specific small interfering RNAs in plants (alone and in combination), that are homologous to only the mRNA of the target insect. In order to express specific siRNAs, we will use an artificial miRNA (amiRNA)-based approach (Liang, He et al. 2012). Artificial miRNAs offer great potential for plant silencing, and offer opportunities to specifically silence targets, even multiple targets (Sablok, Perez-Quintero et al. 2011). We will use standard amiRNA cloning approaches and express single and stacked amiRNAs by two methods: using the 35S promoter and by using a modified begomovirus, the Tomato mottle virus A component. The pToMoVA plasmid is designed as a 1.5 mer copy of the ToMoV A chromosome, which alone is replication competent (Hou 1997). ToMoV replicates in the plant cell nucleus. We will insert the amiRNA backbone sequence into the ToMoV AV1 coding region. Once in the nucleus, the ToMoV A chromosome is released from the plasmid and is replicated (Hou 1997), and thus transcribed amiRNAs will be processed by the plant micro RNA machinery. Both the 35S and ToMoV-based systems will be used first by transient, agroinfiltration (Wang, Turina et al. 2009) and bioassays will be performed using B. cockerelli. We will use small RNA sequencing to ensure that the desired amiRNAs are produced and when constructs that give desired effects are identified these will be used to generate transgenic plants.Objective 3. It also might be possible to engineer insect viruses as vehicles to specifically deliver interfering RNAs to target insects, here our attempt with this strategy is by using the Asian citrus Psyllid, D. citri. Viruses are the most abundant biological entities on earth with estimates as high as 1031 (Breitbart 2005), and they have been reported to occur in essentially all cellular organisms (Koonin, Senkevich et al. 2006, Koonin, Wolf et al. 2009). Insects, including psyllids, are no exception in this regard but surprisingly, the number of viruses reported from psyllids is very small. We collected RNAs from D. citri populations from Brazil, China, Puerto Rico, Florida, California, Taiwan and Pakistan, and next generation sequencing and bioinformatics to identify viruses in D. citri. We discovered 5 new viruses. We are attempting now to clone full-length cloned cDNAs for 3 of them and to engineer them for use in D. citri. We also are using cloned infectious constructs of Flock house virus (FHV) and Cricket paralysis virus (CrPV), and engineering them to target specific RNAs in our insect vectors of choice. We maintain 4 biotypes of D. citri in culture at the CRF and we have aphids in culture in the CEF. We are using the recombinant viruses in attempts to induce systemic RNAi responses in the target insect vectors The intent here is to engineer the recombinant virus to target sequences that may be critical for insect vector, or interfere with its ability to transmit the corresponding plant pathogen. If this approach shows promise, we also will investigate using these viruses to expressed proteins in the corresponding insect vectors.