INVESTIGATOR: Ham, J. H.
PLANT PATHOLOGY & CROP PHYSIOL
LOUISIANA STATE UNIVERSITY
BATON ROUGE, LOUISIANA 70893
MOLECULAR BIOLOGY OF BACTERIAL PANICLE BLIGHT OF RICE AND RICE DEFENSE SYSTEMS FOR THIS DISEASE
CLASSIFICATION HEADINGS: R212 . Pathogens and Nematodes Affecting Plants; S1530 . Rice; F1040 . Molecular biology; S4010 . Bacteria
NON-TECHNICAL SUMMARY: Burkholderia glumae is the chief causative agent causing bacterial panicle blight (BPB) in rice. Outbreaks of BPB have resulted in severe yield losses in the southern United States including Texas, Arkansas and Louisiana. Grain rot, seedling rot and sheath rot of rice, also caused by B. glumae, are growing problems in East and Southeast Asia and Central America. Favored by high temperatures, B. glumae has become a serious threat to rice production in Louisiana and throughout the world due to the current global climate changes. Despite its economic importance, virulence mechanisms of B. glumae are poorly understood compared with that of other important plant pathogenic bacteria because the BPB research is currently in its early stages worldwide. This project aims to comprehensively identify and characterize all the potential virulence factors of B. glumae by mutational analysis, and to investigate how each virulence factor contributes to pathogenesis and affects the host defense system. This project will enable the primary P.I. and co-workers to expand the knowledge of B. glumae virulence factors and their regulatory systems as well as the rice defense systems for this pathogen.
OBJECTIVES: Burkholderia glumae is the major causal agent for bacterial panicle blight (BPB), grain rot and seedling rot in rice. In the southern United States, including Louisiana, BPB is a devastating problem in rice production because of its endemic, and occasionally epidemic, development each year and the significant yield loss that it causes. The high temperatures and humidity normally occurring in Louisiana during the growing season are highly favorable for disease development. The symptoms of BPB include seedling blight, sheath rot and panicle blighting with significant yield losses. Now, BPB is the second most important rice disease in Louisiana after sheath blight (Rhizoctonia solani). In spite of its economic importance, not much has been studied on virulence mechanisms of BPB. The long-term goal of this project is to comprehensively identify and characterize virulence factors of B. glumae and to understand how each of those factors functions in bacterial pathogenesis in either susceptible or resistant rice cultivars. In light of this goal, the specific objectives of this project are the following: 1. To understand the virulence mechanism(s) of Burkholderia glumae, the major causal agent of bacterial panicle blight. 2. To study the molecular basis of rice defenses against B. glumae. a. Differential expression of grain development genes of rice. b. Expression of the defensin-like peptides induced by B. glumae to determine their anti-microbial or insecticidal activities. c. Identification of the elements of B. glumae that induce rice defense genes and innate immunity. Expected outposts: This project will greatly expand our knowledge of the virulence factors of B. glumae, for which only limited numbers of pathogenic determinants have been studied. We will build the basis for further studies on the regulatory system for the virulence factors of B. glumae, which is virtually an unexplored area. This project will not only add new information about virulence factors of B. glumae, but also will allow us to establish an excellent model system for rice-pathogen interactions. Most important is that the knowledge gained from this project can be applied to develop alternative control measures for BPB. For example, avirulent or hypo-virulent B. glumae strains obtained from this project can be used as biological control agents that not only prevail over virulent strains but induce rice defense systems. As BPB is one of the two most important rice diseases in the southern United States, our research project on BPB will have a strong impact on United States rice production. In addition, the study of rice defense responses to B. glumae will add to our knowledge of general rice defense systems and consequently contribute to the study of disease resistance to other major rice diseases such as sheath blight and blast.
APPROACH: 1. Understanding the virulence mechanism of Burkholderia glumae. For this specific objective, a highly virulent strain of B. glumae, 336gr-1, will be randomly mutated with the miniTn5gus transposon. MiniTn5gus can generate transcriptional fusions in mutated genes with the reporter gene gus that encodes beta-glucuronidase (Gus). Thus, the miniTn5gus mutants of B. glumae 336gr-1 can be used for examining the expression of virulence genes as well as their phenotypes in virulence. The miniTn5gus insertion sites of screened mutants can easily be identified by direct sequencing of the mutant genomic DNA using an Epicentre Technologies (Madison, WI) MasterPure DNA purification kit, BigDye terminator (Perkin-Elmer), and specific primers that hybridize to both ends of the transposon. This identification procedure is simple and fast because additional steps for the shotgun cloning of the miniTn5gus flanking regions can be bypassed. 2. Study the molecular basis of the rice defense against B. glumae. Previously, Dr. Rush's group acquired the gene expression profile data from microarray experiments in which the genes for seed-development proteins and defensin-like peptides were specifically induced in the partially resistant cultivar Jupiter in response to B. glumae. These preliminary data will be an important basis of this specific objective. a. Differential expression of grain development genes of rice The grain development genes selected from the previous microarray data will be further confirmed for their induction or suppression levels in response to B. glumae 336gr-1 with reverse transcription polymerase chain reaction (RT-PCR). Using the RT-PCR technique and different bacterial mutants, we will examine what bacterial element(s) are responsible for INDUCTION of the grain-development genes in the partially resistant cultivar Jupiter, or for SUPPRESSION of them in the susceptible cultivar Trenasse. b. Expression of the defensin-like peptides induced by B. glumae to determine their antimicrobial or insecticidal activities The previous gene expression profile data showed that a gene encoding anti-microbial defensin-like peptide was strongly induced by B. glumae in the partially resistant cultivar Jupiter. Thus, it is very probable that the strong induction of the antimicrobial peptide is an effective defense mechanism of rice against the pathogen. To test this hypothesis, the defensin-like peptide will be purified and its anti-microbial and further insecticidal activities will be examined. The cDNA for the defensin-like peptide will be cloned in vectors designed for E. coli or yeast expression systems, and the expressed peptide will be purified using appropriate affinity columns. c. Identification of the elements of B. glumae that induce rice-defense genes and innate immunity. Using the RT-PCR technique and different bacterial mutants, we will examine what bacterial element(s) are responsible for INDUCTION of the grain-development genes in the partially resistant cultivar Jupiter, or for SUPPRESSION of them in the susceptible cultivar Trenasse.
KEYWORDS: burkholderia glumae; bacterial panicle blight of rice
Name: Ham, J. H.