Location: Vegetable Crops Research2013 Annual Report
1a. Objectives (from AD-416):
Objective 1: Identify bacterial pathogen genetic targets for disease management by determining differences in genome-wide gene expression using a field-tested strain of the snap-bean pathogen Pseudomonas syringae grown under a variety of environmental conditions. Objective 2: Analyze replication of Tomato spotted wilt virus in the plant host and thrips vector through quantification of replicative forms of the virion during infection. Objective 3: Quantify the aster yellows phytoplasma load in the leafhopper, determine the relationship of AYp load to successful transmission of disease to the carrot host, and use this information to improve detection of the aster yellows phytoplasma in the insect under field conditions.
1b. Approach (from AD-416):
For Objective 1: Our initial microarray analysis identified approximately 1200 genes that are regulated by GacS/GacA. During this project period, we will analyze the affect of bacterial growth conditions on gene expression including varying pH, iron availability and liquid vs. solid media. High quality RNA will be prepared using our standard bacterial protocol. RNAs will be used to probe commercially available genomic expression arrays containing oligo DNA markers for all 3,840 genes within the B728a genome. The vendor performs standardized hybridization protocols, with the chip data processed by the SY using proprietary software. Changes in gene expression will be confirmed using real-time RT-qPCR. Genes that show differential expression under the various growth conditions will be mutated and their effect on plant virulence determined. For Objective 2: We developed real-time RT-qPCR primers to quantify the total RNA species produced by Tomato spotted wilt virus (TSWV). We will use primers specific to non-coding regions to determine the amount of virion and virion complementary RNA. The mRNAs of TSWV are capped but not poly(A) tailed. We will use methods that specifically enrich capped mRNA to distinguish between the mRNA, virion RNA, and virion-complementary RNA produced by TSWV infection. The viral RNA will be quantified by real-time RT-qPCR using our standard protocols. Expression of TSWV mRNAs during plant infection will be compared to the expression of these RNAs during infection of the insect vector, thrips. The virus is only acquired by 1st instar larval thrips. We will quantify TSWV replication and gene expression in populations of larval and adult thrips. For Objective 3: We will use primers to two aster yellows phytoplasma (AYp) gene sequences and an aster leafhopper (ALH) gene sequence as a target for amplification of the aster leafhopper chromosomal DNA. The presence of phytoplasma in plant and insect tissue extracts will be detected using traditional PCR or nested PCR reactions. AYp copies per insect will be determined with both AYp specific primer pairs using real-time qPCR. We will also determine AYp copies per cp6 chromosomal marker to access the utility of using a chromosomal marker instead of copies per insect as standardization. Our objectives are to measure the increase of AYp copy number in ALH over time, examine AYp copy number differences between male and female insects, and determine the phytoplasma levels required for successful AYp transmission. For transmission analysis, ALH will be given a 48-hour acquisition access period on an AYp-infected Chinese aster (Callistephus chinensis) followed by transfer to rye seedlings to allow for the propagation of the phytoplasma within the leafhoppers. Leafhopper individuals will be clipped to healthy aster plants and given a 24-hour inoculation access period (IAP). Disease will be assessed visually on individual aster plants at 20 and 30 days post-IAP and aster petioles will be assayed for the presence of AYp by nested PCR. A positive detection in the aster plant will relate directly to a positive transmission by an ALH.
3. Progress Report:
For objective 1, we analyzed the expression and function of genes involved in disease on bean. The bacterium Pseudomonas syringae pv. syringae causes brown spot disease of snap bean and is an agronomically important pathogen in Wisconsin and other snap-bean producing areas of the United States. Our microarray analysis combined with mutational analysis of the bacterium identified the toxins syringopeptin and syringolin A as major contributors to brown spot disease development on bean. This finding establishes that breeding toxin resistance plants could provide a novel control method for brown spot disease of snap bean. We have completed the microarray analysis of gene expression in B728a grown on solid media and in liquid media (planktonic growth). The results are very similar to what we discovered on solid media. The few differences in gene expression noted are currently being genetically analyzed to determine the importance of the target genes for planktonic growth. For objective 2, we developed methods to analyze viral replication within infected plants and insect vectors. Our target viruses are Maize fine streak virus and Tomato spotted wilt virus (TSWV). Maize fine streak virus is an important pathogen of corn in the United States while TSWV infects many agronomically important crops including tomato, lettuce, and pineapple. Unlike current methods of detection, our methods can distinguish between active viral infections and the mere presence of the virus in infected tissues. We have completed our analysis of Maize fine streak virus using two approaches for quantifying the viral gene expression. We established that expression of two viral genes was elevated early in the infection process. This result demonstrates that plant infecting Rhabdoviruses have a gene expression strategy that is significantly different from animal infecting Rhabdoviruses. This finding provides genetic targets for development of plant resistance. For objective 3, we developed a quantitative assay to measure aster yellows phytoplasma amounts within the aster leafhopper insect vector. Aster yellows is a major disease problem in vegetables such as carrot. Conducting time course experiments tested this method. Average aster yellows phytoplasma amounts within insects was measured in leafhoppers and copies per insect ranged from 3.4 thousand to 1.8 million occurring at 1 and 7 days. Aster yellows phytoplasma numbers per insect increased approximately 100-fold in insects that successfully acquired the pathogen. This method will improve our ability to study biological factors governing aster yellows phytoplasma replication in the leafhopper and determine if aster yellows phytoplasma amount within an insect is associated with frequency of disease transmission.
Hogan, C.S., Mole, B.M., Grant, S.R., Willis, D.K., Charkowski, A.0. 2013. The type III secreted effector DspE is required early in Solanum tuberosum leaf infection by Pectobacterium carotovorum to elicit cell death, and requires Wx(3-6)D/E motifs. PLoS One. Available: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0065534.