Influence of Potato Leafroll Virus on Myzus Persicae-Potato Interactions

<p>NON-TECHNICAL SUMMARY:<br/> Most pathogens depend on vectors for transmission among host plants and, ultimately, for survival. Thus, vector behavior can affect pathogen epidemiology. Potato leafroll virus (PLRV), which is primarily transmitted by the potato-peach aphid (Myzus persicae), causes one of the most serious diseases of potato (Solanum tuberosum) worldwide, reducing crop yield by 40-70%. The objectives of my proposed research are to investigate the molecular mechanisms that underpin potato-M. persicae-PLRV interactions and determine the functions of specific PLRV proteins in vector-plant relationships and virus epidemiology. By studying potato gene expression, metabolic and protein responses to PLRV and M. persicae, I will test the hypothesis that PLRV-induced changes increase nutrient availability for aphids and suppress potato defense responses. In addition, I
will clone and express each of the nine PLRV proteins in transgenic potatoes and tobacco. Using insect bioassays and biochemical screens, I will determine the impact of individual PLRV proteins on plant-aphid interactions. My proposed research will promote the development of novel virus-vector control strategies in crop plants, thus leading to reduced pesticide use and increased food safety. By studying the mechanisms mediating plant-virus-vector interactions, I will address the following program challenge and foundational areas: (1) Plant health and production and plant products; (2) Food safety, nutrition, and health; (3) Agriculture systems and technology; and (4) Food security.
<p>APPROACH:<br/> Hypothesis 1.1: Phloem-specific changes in gene expression differ between healthy and infected potatoes, with and without aphid feeding. Description and methods. As M. persicae are more fecund on PLRV-infected potatoes [30, 38-41], it is likely that changes in the plant transcriptional environment underlie at least some of the difference in insect performance. To study these differences, I will sequence cDNA libraries generated from healthy and PLRV-infected potatoes with and without aphid feeding using Illumina sequencing technology (RNAseq). These data will identify candidate genes and mechanism underling the function of viruses in plant-vector interactions. I will validate changes at the protein level using iTRAQ technology, a discovery-based proteomics approach, and selected reaction monitoring (SRM) mass spectrometry in collaboration with the Cilia
lab. Expected outcomes and measures. These experiments will provide quantitative data about gene expression and protiens in potatoes during virus infection and aphid feeding. These studies are likely to confirm my hypotheses that there are specific gene expression changes in virus infected potatoes that underlie the extended phenotype of enhanced aphid performance. I expect mRNA changes to be consistent with protein changes. However differences may be found between mRNA and corresponding protein targets, as many regulatory mechanisms operate at the protein level. Gene expression changes that I will identify may indicate that primary or secondary metabolism is being reprogrammed by PLRV infection. These results will help to guide experiments described below (Hypothesis 1.2), where I will investigate changes in host plant nutritive value and defense responses that can affect aphid feeding
and reproduction. Hypothesis 1.2: Increased palatability of virus infected tissue increases aphid performance. Description and methods. PLRV-induced changes in potato metabolism may alter plant palatability for aphids and could explain the observed increases in aphid reproduction on virus-infected plants [40, 61]. Both increased nutrient content and decreased defenses in the host plant could increase aphid performance. I will conduct experiments to determine whether PLRV alterations in plant metabolism contribute to increased palatability for aphids. Outlined below are some examples of nutrient changes and defenses I will examine. However, this research will be guided by results from Hypothesis 1.1 and specific focus may change accordingly. Expected outcomes and measures. If the plant palatability of healthy and PLRV-infected tissue differs for aphids, then I expect to see differences
between free amino acid content and/or plant defense responses among the treatments. I expect free amino acids to be elevated in infected plant tissue and for aphids to acquire greater amounts of this nutrient in their body. I also expect aphid defenses to be reduced in potatoes. Together, these experiments will determine whether PLRV infection re-programs potato metabolism and thereby increases the palatability of the tissue for aphids. However these results are not mutually exclusive, one, all or a combination of these expected results may be found and contribute to enhanced aphid growth and fecundity. Hypothesis 2.1: Phloem expression of specific PLRV proteins increases aphid reproduction. Description and methods. I hypothesize that activity of one or more PLRV proteins contributes to changes in the host plant that influence aphid performance. To test this, I will clone the coding
sequence of the 9 mature PLRV proteins [53, 83]into a derivative of the Gateway-compatible vector pMDC85 which is available in the Jander lab [84]. Transgene expression in pMDC85 has been modified to be under the control of the phloem-specific promoter, AtSuc2 [85]. In addition, the pMDC85 vector attaches a GFP and HIS-tag [84], opening the possibility for doing protein purifications, immunoprecipitation (IP) and western blot experiments based on epitope tags. Expected outcomes and measures. I expect expression of one or more PLRV proteins will increase aphid fecundity, similar to the actual virus infection. I expect this will correlate with changes in plant metabolism in virus infected tissue identified in Hypothesis 1.2. If changes in aphid fecundity are observed in PLRV diet experiments I expect individual proteins may also impact aphid fecundity through diet. Hypothesis 2.2: PLRV
proteins expression determines volatile emissions and epidemiology. Description and methods. Potato plants infected withPLRV attract and arrest M. persicae more strongly than non-infected plants [30, 87]. These differences were due to altered volatile emission in virus infected host plants [39, 88]. Because insect preference and emigration rate from infected plants will ultimately determine vector inoculation and future transmission, I will determine if expression of PLRV proteins in the host plant will alter aphid attraction/ arrestment. Expected outcomes and measures. I expect that expression of one or more PLRV proteins will increase volatile production and aphid attraction/arrestment on plants, similar to actual virus infection. However it is likely that multiple PLRV proteins alter plant-vector dynamics. For instance, aphid arrestment on PLRV-infected leaves changes as disease
progresses [41]. The induction of a different suite of volatiles may be involved in these changes. If single proteins do not produce significant differences, I expect a combination of PLRV proteins may be involved and this will be examined using transient expression in N. benthamiana.