2012 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.
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. The Gac pathway genes encode regulators that are required for disease on snap bean in the field. We established that the Gac transcriptome in B725a includes over 1200 genes regulated by Gac. Our microarray analysis combined with mutational analysis of the bacterium identified the toxins syringopeptin and syringolin A as major contributors to brown spot disease symptom development on bean. This result establishes that breeding toxin resistance plants could provide a novel control method for brown spot disease of snap bean. We are now analyzing gene expression under a variety of growth conditions. We have completed preparation of ribonucleic acids (RNA) samples from B728a grown on solid media and in liquid media (planktonic growth) and that RNA is currently being analyzed by microarray by the vendor.
We improved methods to analyze viral replication of Maize fine streak virus (MFSV) and Tomato spotted wilt virus (TSWV) within infected plants. MFSV 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 developed novel methods for isolating polymerase chain reaction (PCR) fragments for cloning and use directly as standard curves of real-time real-time reverse transcription polymerase chain reaction (RT-qPCR) analysis. We constructed clones containing viral sequences for use as standards for measuring the viral RNA levels for both MFSV and TSWV. We confirmed the over-expression of two plant virus-specific transcripts from MFSV within infected corn and showed that expression was elevated early in the infection process. These two MFSV transcripts are now prime targets for transgenic virus resistance.
The aster yellows phytoplasma (AYp) is transmitted by the aster leafhopper (ALH), Macrosteles quadrilineatus Forbes, in a persistent and propagative manner. To study AYp replication and examine the variability of AYp titer in individual ALHs, we developed a quantitative real-time polymerase chain reaction (qPCR) assay to measure AYp concentration in insect deoxyribonucleic acid (DNA) extracts. AYp titers per insect and relative to an ALH chromosomal reference gene, cp6 wingless (cp6), increased approximately 100-fold in insects that acquired the AYp. This method will improve our ability to study biological factors governing AYp replication in the ALH and determine if AYp titer is associated with frequency of transmission.
Quantification of the aster yellows phytoplasma in the leafhopper insect vector. The aster yellows phytoplasma causes yield and quality losses in a wide range of plant species including many common vegetables, ornamental, and agronomically important field crops such as carrots. ARS scientists in collaboration with researchers in the Department of Entomology at the University of Wisconsin-Madison developed a quantitative assay to measure aster yellows phytoplasma numbers within the insect. The amount of aster yellows phytoplasma varied widely among individual leafhoppers and the amount of the aster yellows phytoplasma increased approximately 100-fold within infected insects within one week of feeding on infected plants. This establishes that the aster yellows phytoplasma replicates quite effectively within the insect vector and explains why leafhoppers are such a good vector for spread of the aster yellows disease. This method will determine the level of aster yellows phytoplasma necessary to transmit the disease and this information will be used to improve aster yellows treatment models used for pesticide application in the field.