2010 Annual Report
1a.Objectives (from AD-416)
The long-term objective of this project is to develop an improved understanding of the genetics of bacterial and viral pathogens that cause disease on snap bean, tomato and potato. Over the next 5 years we will focus on the following objectives:
Objective 1: Use P. syringae pv. syringae B728a genomic expression chips to identify and characterize genes regulated by the gacS/gacA two-component regulatory system. Sub-objective 1.A. Use genomic expression chips to identify the members of the gacA/gacS transcriptome that are regulated under a variety of growth conditions. Sub-objective 1.B. Functional genomic analysis of gacS/gacA regulated genes.
Objective 2: Develop and analyze transgenic plants expressing a viral protein that may inhibit Tomato spotted wilt virus (TSWV) transmission by thrips.
Sub-objective 2.A. Develop real-time RT-PCR methodologies to quantitate TSWV replication in host plants and the thrips vector. Sub-objective 2.b. Construct and characterize transgenic tomato plants expressing the TSWV glycoprotein GN-S.
1b.Approach (from AD-416)
For Objective 1: Bacterial growth conditions that will be analyzed include varying pH, iron availability and liquid vs. solid media. These growth conditions are all known to affect the growth of bacteria on plants. High quality RNA will be prepared using standard bacterial protocols. RNAs will be used to probe commercially available genomic expression arrays containing oligo DNA markers for all 3,840 genes within the B728a genome. Reproducibility will be ensured by having standardized hybridization protocols performed by the vendor, with the chip data processed by the SY using proprietary software. Changes in gene expression will be confirmed using real-time RT-PCR. Genes that show differential expression under the various growth conditions will be mutated and their effect on plant virulence determined.
For Objective 2: All three TSWV RNA contain very similar but not identical sequences at their ends. We will use these end sequences to design primers that are specific to either the genomic RNA (contained in the viral particle) or anti-genomicRNA (necessary for replication) to produce cDNA specific to that RNA. We will determine the amounts of viral message RNA species by using random hexamers to generate cDNA. The viral RNA within each cDNA will be quantitated by real-time PCR using our standard protocols. The amount of each RNA species will be determined by using a standard curve consisting of a dilution series of cloned viral DNA of known concentration. As a preliminary to the analsysis of TSWV, we will determine the relative amounts of genomic, anti-genomic, and viral mRNAs expressed by the maize pathogen Maize fine streak virus. MFSV is a mono-partite negative-sense virus that contains only a single RNA genome and avoids the complexity of distinquishing three RNA genomes containing related sequences as is the case with TSWV.
We have shown that feeding thrips a modified form of the TSWV glycoprotein GN (designated GN-S) dramatically inhibits the acquisition of the virus and the ability of the thrips to transmit the virus. This most likely is due to the saturation of viral binding sites within the thrips guts by GN-S thus preventing viral binding and transport of the TSWV virion through the intestinal lining. We will express the GN-S protein in potato and other hosts to establish that this protein can inhibit the acquisition and transmission of TSWV when expressed within the plant. The GN-S ORF will be cloned into an Agrobacterium vector. This construct will be either transiently expressed using an Agro launching technique or transformed into a susceptible host. Plants will be analyzed for GN-S gene expression using real-time RT-PCR and GN-S protein expression by western blot. Thrips will be fed on transiently expressing leaf discs or transformed plants showing a high level of expression of the GN-S protein for a two hour acquisition period and then moved to TSWV infected hosts. Acquisition of TSWV by thrips will be analyzed using real-time RT-PCR and transmission of TSWV to host plants will be quantitated using a leaf disc or green house assay.
The GacS-GacA genetic regulon controls the expression of a variety of important virulence functions in the snap bean pathogen Pseudomonas syringae pv. syringae B728a, to the degree that mutation of either the gacS or the gacA gene results in a complete loss of pathogenicity. Microarray analysis demonstrated that 21% of the total genes (1100 of 5137 predicted genes) of P. s. syringae strain B728a was controlled by gacA/gacS. A bacterial strain containing a mutation in the gacS/gacA-regulated salA gene shows a similar expression profile to the B728a parent, except in gene regions involved in the production of three bacterial toxins. Since we previously established that salA mutants are unable to cause disease symptoms on bean, this implicates these three toxins as significant contributors to the disease process. Recent experiments indicated that bacterial swarming, a phenotype that has been associated with virulence and is also regulated by the Gac regulon, does not require bacterial growth. We identified fundamental differences in gene expression between liquid grown bacteria and bacteria grown in colonies on solid media. Both swarming and micro-colony formation are expected to be contributors to bacterial fitness on plants.
We successfully quantified the Tomato spotted wilt virus (TSWV) transcripts encoding the nucleocapsid protein (N) and the non-structural protein (NSs) within infected host plants using real-time reverse transcription polymerase chain reaction (RT-qPCR.) High efficiency primers specific for these messenger ribonucleic acids (RNAs) were developed and used for the experiments. Our results indicate the N gene is expressed approximately 5-fold higher than the NSs gene in infected plants. This result set up the analysis of the expression of these two genes and the remaining three TSWV genes within Western Flower Thrips, the agronomically important vector of TSWV. Our efforts will confirm that TSWV infects and actively replicates within the insect vector. This information is vital for formulating novel control strategies for the acquisition & spread of this virus.
We improved methods to analyze RNA produced by Maize fine streak virus (MFSV) within infected maize tissue using real-time RT-qPCR. We established that MFSV virion RNA is approximately 50-fold more abundant than the replicative virion-complementary RNA in symptomatic maize leaf tissue. The abundance of the MFSV gene transcripts decreased with distance from the 3´ promoter suggesting transcription in infected maize similar to transcription in the well studied rhabdovirus Vesticular stomatitis virus (VSV). Our research discovered that two MFSV specific transcripts, not present in VSV or other animal Rhabdoviruses, accumulated to higher levels than predicted suggesting that these transcripts are produced by an alternate transcription process or exhibit increased stability relative the remaining MFSV transcripts. This result not only illustrates differences in the expression of plant-infecting Rhabdoviruses compared to their animal-infecting relatives, but also provides potential targets for inhibition of the replication of MFSV in its agronomically important host, corn.
Quantification of the aster yellows phytoplasma within its insect vector. The aster yellows phytoplasma is transmitted by the aster leafhopper, or Macrosteles quadrilineatus. Aster yellows is an agronomically important pathogen of carrot. ARS scientists in the Vegetable Crops Research Unit in collaboration with university researchers in the Department of Entomology at the University of Wisconsin-Madison developed a quantitative real-time reverse transcription polymerase chain reaction PCR (qPCR) assay to measure aster yellows concentration in insect deoxyribonucleic acid (DNA) extracts. Our results demonstrated that the aster yellows phytoplasma efficiently replicates within the leafhopper reaching a maximum titer in 10 days. This new technique will enable us to examine the biological factors governing aster yellows replication in the leafhopper and examine if aster yellows population size is associated with the frequency of transmission.
Determination of the regulation of ß-amylase within high and low activity malting barley. ß-amylase is an enzyme important to the malting and brewing industry. The objective of this study was to determine the genetic basis of differences ß-amylase activity in barley (Hordeum vulgare L.). ARS scientists in the Vegetable Crops Research Unit and the Cereal Crops Research Unit in collaboration with university researchers in the Department of Agronomy at the University of Wisconsin used real-time RT-qPCR and protein activity analysis to investigate basis for differing ß-amylase activities among four barley cultivars. Our results demonstrated that cultivars with high ß-amylase activity also had higher messenger ribonucleic acid (mRNA) levels suggesting increased gene expression is the source of the increased ß-amylase activity. This increase was not linked to known genetic markers within the ß-amylase gene. We also found that total protein was higher in grains from high ß-amylase cultivars. Higher levels of total protein could also contribute to the higher levels of ß-amylase activity.
Identification of molecular targets for juvenile hormone action in insects. The insect juvenile hormones represent a family of molecules that regulate a diversity of processes in the insect life cycle. Juvenile hormone affects insect development by maintaining the larval stage and inhibiting metamorphosis, and it is this feature that has led to the development of juvenile hormone analogs and agonists as agronomically important insecticides. ARS scientists in the Vegetable Crops Research Unit in collaboration with university researchers in the Department of Entomology at the University of Wisconsin-Madison used microarray analysis to identify specific genes influenced by juvenile hormone. Our microarray analysis revealed relatively few insect genes were verifiably altered in their expression by addition of juvenile hormone. Two genes that showed increased expression were of unknown function. Expression of the Epac gene involved in insect development was increased four-fold in the presence of juvenile hormone. Identification of Epac and additional juvenile hormone influenced genes provide novel targets for genetic and chemical control of insect pests.
Willis, D.K., Wang, J., Stanford, J.R., Orth, A., Goodman, W.G. 2010. Microarray Analysis of Juvenile Hormone Response in Drosophila melanogaster S2 cells. Journal of Insect Science. 10(6). Available: http://www.insectscience.org/10.66/i1536-2442-10-66.pdf.