Progress 01/01/24 to 12/31/24
Outputs
Target Audience:Scientists in agricultural sciences and microbiology, graduate students and post-docts in related fields, researchers and engineers in ag industry as well as public and private institutions, and stakeholders of rice industry including growers. Changes/Problems:Unexpectedly, we identified new rice pathogens infecting panicles like B. glumae while working on this project. We will complete reporting this finding of new rice pathogens through close communication with Louisiana Department of Agriculture and Forest (LDAF) and USDA APHIS. What opportunities for training and professional development has the project provided?Four graduate students (two Ph.D. and one master students major in plant pathology, and one Ph.D. student in biology) and one undergraduate student have been trained for skillsets in molecular biology, microbiology, biochemistry, and bioinformatics, which have been employed for this project. These graduate and undergraduate students had chance to attend and give oral or poster presentations at regional, national, and international conferences, which were 1) the 2024 American Phytopathological Society Southern Division Meeting (Columbia, South Carolina), 2) the American Phytopathological Society Annual Meeting (Plant Health 2024, Memphis, Tennessee), 3) the International Temperate Rice Conference (New Orleans, Louisiana), and 4) the Corteva Plant Science Symposium (Baton Rouge, Louisiana). How have the results been disseminated to communities of interest?The scientific results obtained directly and indirectly from research activities for this project were presented at the 2024 American Phytopathological Society Southern Division Meeting (Columbia, South Carolina), the American Phytopathological Society Annual Meeting (Plant Health 2024, Memphis, Tennessee), the International Temperate Rice Conference (New Orleans, Louisiana), and the Corteva Plant Science Symposium (Baton Rouge, Louisiana). Our research program including this project was also introduced to rice growers and stakeholders at the Louisiana Rice Field Day (H. Rouse Caffey Rice Research Station, Rayne, Louisiana) and the summer outreach program for K-12 students and science teachers (LSU campus, Baton Rouge, Louisiana). What do you plan to do during the next reporting period to accomplish the goals?1. Characterize the signaling and regulatory network connected with qsmR Transcriptome profiles of two virulent (336gr-1 and 411gr-6) and one naturally avirulent (257sh-1) strains will be analyzed in their wild type, qsmR-deficient, and quorum-sensing-deficient backgrounds. This work will include analysis of differential gene expression and co-expression network. Non-coding RNAs will also be investigated for post-transcriptional regulation by qsmR and quorum-sensing genes. ChIP-seq assay will be performed using the experimental systems developed in this project year to identify genes regulated by physical binding of QsmR regulatory protein. Functions of qsmR gene on cell cycle will be further studied based on our observation of abnormal cell elongation by qsmR mutation. 2. Identify metabolic conditions and signals affecting the function of qsmR and virulence Different carbon sources and putative QsmR-binding molecules will be tested for their effects on bacterial growth and pathogenicity. Putative QsmR-binding molecules will be tested for their physical binding to the purified QsmR protein using the surface plasmon resonance (SPR) or the nuclear magnetic resonance (NMR) technique. 3. Investigate the dynamics of qsmR function associated with bacterial adaptation to changing environments. Spontaneous avirulent mutants isolated from bacterial culture and infected plants will be characterized for mutation in two foci: one on mutation patterns in qsmR gene and the other on changes in genome structure and mutations in other genes. Morphology of spontaneous mutants will be investigated in both cellular and colony levels.
Impacts
What was accomplished under these goals?
The major goal of this project is to understand the function of qsmR gene in the rice pathogenic bacterium Burkholderia glumae, as an essential regulatory element for pathogenicity. 1. Characterize the signaling and regulatory network connected with qsmR We investigated the functional relationship between qsmR and the quorum-sensing system, another key signal and regulatory element for the virulence of B. glumae. We found that qsmR was not influenced by the quorum-sensing genes, tofI and tofR, in its expression. qsmR gene only partially affected the expression of the two quorum-sensing genes, suggesting that qsmR gene and the quorum-sensing genes exert their regulatory functions independently to each other with only a partial influence of qsmR on the quorum-sensing genes. We also found that tepR gene, a previously identified regulatory gene that suppresses the virulence of B. glumae, was inhibited by qsmR gene as well as the quorum-sensing genes. Additionally, we identified hfq gene encoding a small RNA-binding protein as an important regulatory factor for expression of qsmR and the quorum-sensing genes. With these new research findings, we could understand the regulatory hierarchy among the crucial regulatory elements that determines the pathogenic behaviors of B. glumae. Another accomplishment achieved while conducting experiments for this project was observation of cell elongation by qsmR mutation, which strongly suggests that qsmR is also required for proper cell division. This abnormal cell morphology caused by qsmR will be continued next project year. 2. Identify metabolic conditions and signals affecting the function of qsmR and virulence Chemical compounds predicted to bind to QsmR protein based on computer modeling were tested for their effects on the virulence of B. glumae. After identification of three chemical compounds that enhance the virulence (indicated by the production of phytotoxin), additional six compounds selected based on predicted binding affinity were tested. Among these, four compounds (malic acid, deoxycolic acid, glutamic acid, guanine) showed 20 - 30% reduction in phytotoxin (toxoflavin) production at the concentration of 1-10 µM. 3. Investigate the dynamics of qsmR function associated with bacterial adaptation to changing environments Spontaneous mutants of B. glumae were screened based on abnormal colony morphology and their lost virulence function was confirmed. It was discovered that most of them have about 300 base pair deletion in qsmR gene. Additional spontaneous mutants are being screened from infected rice plants. Occurrence frequency of spontaneous mutants was determined at different growth stages of bacterial culture. Moreover, five naturally avirulent strains of B. glumae were characterized for their whole genome DNA sequences. For more biochemical study on qsmR and its encoded protein QsmR, experimental system for expression and detection of QsmR protein, including Western blot, was established. With this experimental system, expression of both QsmR and QsmR-His (QsmR protein tagged with multiple histidine residues) in B. glumae was confirmed. Another accomplishment achieved unexpectedly while conducting this project was identification of new bacterial pathogens from rice samples, which showed symptoms on panicles similar to bacterial panicle blight. From this study, two manuscripts have been prepared of which one has been accepted for publication in Plant Disease.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Lelis TDP, Bruno J, Padilla J, Barphagha I, Ontoy J, and Ham JH (2024) qsmR encoding an IclR-family transcriptional factor is a core pathogenic determinant of Burkholderia glumae beyond the acyl-homoserine lactone-mediated quorum-sensing system. PLoS Pathogens 20(10): e1011862.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Bruno J and Ham JH (2024) Seed priming for defense priming: An innovative approach to enhance the resilience of crop plants to biotic and abiotic stresses. Plant Health Progress 25:228-231.
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Progress 01/01/23 to 12/31/23
Outputs
Target Audience:Scientists in agricultural sciences and microbiology, graduate students and post-docts in related fields, researchers and engineers in ag industry as well as public and private institutions, and stakeholders of rice industry including growers. Changes/Problems:Study on the newly identified chemical compounds, which werepredicted to interact with QsmR proteinfrom a collaborative work with Dr. Brylinski, will be inlcuded as a part of Objective 2. Function of QsmR on the competition with another rice-inhabiting bacterium will also be investigated as parts of Objective 3 to understand itsbroader role inparasitic fitness. What opportunities for training and professional development has the project provided?Threegraduate students (one Ph.D. and one master student major in plant pathology, and one Ph.D. student in biology) have been trained for molecular biological, biochemistry and genomic studies of bacterial plant pathogens through conducting research activities for this project. These graduate studentsalso had chance to attend and make presentations at a national meeting (the American Phytopathological Society Annual Meeting (Plant Health 2023) held in Denver, CO) and professional meetings held in LSU (the Corteva Plant Science Symposium and the LSU/SU AgCenter Joint Annual Conference). In addition, oneundergraduate student (major in biochemistry)hashad been trainedthrough participating in this project, and he had chance to present his research at the Corteva Plant Science Symposium. How have the results been disseminated to communities of interest?The results from this project were presented at the American Phytopathological Society Annual Meeting (Plant Health 2023, Denver, CO),the Corteva Plant Science Symposium (LSU, Baton Rouge, LA), the LSU/SU AgCenter Joint Annual Conference (LSU, Baton Rouge, LA), and the LSU Bacterial Pathogenesis and Host Response Group Meeting (LSU,. Baton Rouge, LA). Our research programon plant bacteria including this project was published in the magazine Futurum (www.futurumcareers.com), which introduces STEM research programs to K9 - 12 students around the world (Issue 22). What do you plan to do during the next reporting period to accomplish the goals?1. Characterize the signaling and regulatory network connected withqsmR Functional relationships between qsmR and the QS system will be characterized through genetic and transcriptomic analysis. The whole genome sequences of the five natural avirulent strains sequenced this year will be characterized further in comparison with the previously characterized avirulent strain 257sh-1 andvirulent strainsto understand the genome architecture and dynamics associated with the virulence trait. One manuscript will be prepared from this work. The spontaneous mutant strains ofB. glumaeisolated from the bacterial culture and infected plants will also be characterized like the natural avirulent strains. Function of qsmR in competition with another rice-inhabiting (but not pathogenic) bacterium will be investigated for more comprehensive understanding of the gene in host plant. 2. Identify metabolic conditions and signals affecting the function ofqsmRand virulence The study on the effect of various plant metabolites (e.g., glucose, fructose and ascorbic acid) and bacterial signals (e.g., quorum-sensing signals) on the expression of qsmR and the virulence trait of B. glumae will be completed. Protein structure of QsmR will be studied more comprehensively in collaboration with Dr. Brylinski (structural biologist in the School of Biological Sciences) to identify more chemicals predicted to interact with the protein and to find clues to engineer new chemicals that can disable QsmR function for virulence. 3. Investigate the dynamics ofqsmRfunction associated with bacterial adaptation to changing environments Natural avirulent strains andspontaneously generated avirulent strains will be tested for their ability to regain virulence in freshly infected rice plants and for their mutations on qsmR. To investigate the patterns of spontaneous mutations in qsmR among the B. glumae population under stress condition or heavily infected plants, PCR amplicons for the qsmR gene will be obtained from the given bacterial population and analyzed for mutation patterns of qsmR in the population using a next generation sequencing, such as Illumina Miseq.
Impacts
What was accomplished under these goals?
The main goal of this project is to elucidate howqsmR, a key regulatory element that determines bacterial pathogenicity of the rice pathogen Burkholderia glumae, controls the pathogenic behaviors ofB. glumaein different infection stages and environmental conditions. 1. Characterization ofthe signaling and regulatory network connected withqsmR We characterized the differential functions of the qsmR gene and the quorum-sensing (QS) system, another key element determining bacterial pathogenicity, among different strains of Burkholderia glumae to determine the functional hierarchy between the two major signaling systems for the virulence. For this,we performed genetic experiments with 20strains of the pathogen from different locations of Louisiana rice fields by generating mutants defective in the qsmR gene and the QS system and subsequent characterization of their pathogenic traits. We also performedgenomics study through high-throughput sequencing to characterize the bacterial genes dependent on qsmR and QS. From our study this year, which expanded the genetic tests to 20natural strains representing different lineages of the pathogen, we could prove our hyothesisthat qsmR is an essential element for pathogenicity in all lineages tested. At the same time, we could prove our hypothesisthatthe QS is essential only for a specific lineage. Moreover, we discovered that qsmR and the QS system function independently, which is a new finding thatchanges the currentmodeland provide important clues for disabling pathogenic ability of this bacterium and other related plant pathogens. 2. Identify metabolic conditions and signals affecting the function ofqsmRand virulence We conducted several rounds of experiments to investigate the effects of vatrious metablic compounds as a sole carbon sourceon the virulence funciton of B. glumae. Tested materials included polygalactunonic acid, ascobic acid, galactose, glucose, and mannose. However, we could not observe any significant effects of those tested materials in both minimal and rice medium conditions. This study is beingcontinuedwith additional carbon source mateirals and different mediumconditions. An exciting progress related to this objective was the identification ofpotential chemical compounds that bind to the QsmR protein based on protein modeling. This achievement was made in collaboration with a structural biology laboratory (Dr. Brylinski) in the Departmentof Biological Sciences. In preliminary experiments, three chemical compounds predicted to bind to QsmR increasedthe prduction of the major virulenc factor toxoflavinbyB. glumae.This breakthrough warrants further studies to identify or engineer new chemical materials that can inhibitthe function of QsmR transcriptional factor encoded by qsmRgene, which will lead to the disabling of the pathogenic ability of B. glumae and of related pathogens. Nevertehless, the biocheminal tests to prove physical binding of the candidate complounds to QsmR protein have been delayed due to the formation ofinclusion body of the protein when it is overexpressed. To solve this issue,we have established aprotocol forthe solublization ofQsmR protein. 3. Investigate the dynamics ofqsmRfunction associated with bacterial adaptation to changing environments Our hypothesis on this objective was that qsmR loses and regains its function easily depending on environmental conditions through spontaneous mutations, rather than regulation of gene expression. Inour experiments this year, we observed that about 20% of bacterial population lost the ability to produce virulence factors (i.e. toxoflavin and extracellular protease) under a prolonged culturecondition. However, many of those bacterial cells defective in the production of thevirulence factors regained their virulence when they were tested on onion bulb scales (the surrogate virulence assay system used in our laboratory for B. glumae). We also conducted similar experiments, in which rice plants were infected with a virulent strain ofB. glumae, andthe bacterial cells were resolated from the infected plants at different infection stages. Theavirulent mutants obtained from bacteiral cultures and infected rice plants (> 10 siolates) have been preserved and will be characterized to identify the cause of the lost virulence.This research will provide valuable information about how B. glumae adapts to changing environments switching between virulent and avirulent modes for long-term survival. Related to this objective, we also created B. glumae cells expressing the green fluorescent protein and the red fluorescent protein, which can be utilized for monitoring bacterial behaviors in plantusing fluorescent microscopy. Meanwhile, we tried to collect more strains of B. glumae including naturally avirulent ones from Louisiana rice fieldsthis year, but only a few new strains of B. glumae could be obtained without any avirulent isolates. Instead, we unexpectedly isolated and identified otherbacterial pathogens from rice plants showing panicle blight symtoms. These bacterial pathogens have not been reported in Louisiana. Accordingly, we are currently in the process of reporting thesenew bacteiral pathogens causing rice panicle blight in Louisiana.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Lelis, T., J. Bruno, J. Padilla, I. Barphagha, and J. H. Ham. 2023. qsmR encoding an IclR-family transcriptional factor is a core pathogenic determinant of Burkholderia glumae beyond the acyl-homoserine lactone-mediated quorum-sensing system. bioRxiv DOI: https://doi.org/10.1101/2023.12.05.570247Phytopathology 112:S3.118. (This manusciprt is under review for publication in PLOS Pathogens).
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Iqbal, A., G. Nwokocha, V. Tiwari, I. K. Barphagha, A. Grove, J. H. Ham, and W. T. Doerrler. 2023. A membrane protein of the rice pathogen Burkholderia glumae required for oxalic acid secretion and quorum-sensing. Molecular Plant Pathology DOI: 10.1111/mpp.13376.
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Progress 01/01/22 to 12/31/22
Outputs
Target Audience:Scientists and graduate students in the field of plant pathology and microbiology,rice growers,county agents,researchers anddevelopersof agribiocompanies, and agricultural consultants. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate students in the Ph.D. program were trained for designing and conducting research projects in genetics, genomics and transcriptomics of bacterial pathogens and plant-microbe interactions. Two undergraduate students majoring in biochemistry and bioengineering, respectively, were trained for basic laboratory skills of microbiology and plant science studies. The graduate students involved in this project could attend and make presentation at the 2022 annual meeting of the American Phytopathological Society (Plant Health 2022, Pittsburgh, PA). How have the results been disseminated to communities of interest?The research outcome obtained from this project was presented at the 2022 annual meeting of the American Phytopathological Society (Plant Health 2022, Pittsburgh, PA) and at the Mississippi State University (Dept. Biochemistry, Molecular Biology, Entomology & Plant Pathology). What do you plan to do during the next reporting period to accomplish the goals?1) Characterize the signaling and regulatory network connected withqsmR Comparative whole genome sequence analysis will be performed with the naturally avirulent strains of B. glumae to identify genomic features associated with the avirulent trait. Functional interactions between the qsmR gene and other regulatory genes and signaling systems will be characterized. 2) Identify metabolic conditions and signals affecting the function ofqsmRand virulence Effects of various carbon sources and signal molecules on the virulence and the expression of qsmR will be characterized. 3)Investigate the dynamics ofqsmRfunction associated with bacterial adaptation to changing environments Changes in pathogenic traits and accompanying qsmR function will be examined throughout a disease cycle and in different environmental conditions though targeted and/or whole genome sequencing.
Impacts
What was accomplished under these goals?
1) Characterize the signaling and regulatory network connected withqsmR ? To characterize the differential regulons of qsmR in three different strains of Burkholderia glumae, RNA-seq experiments were conducted with qsmR-deleted mutant derivatives of three strains, 336gr-1 (virulent strain) and 411gr-6 (hypervirulent strain). These experiments also included the mutant derivatives of 336gr-1 and 411gr-6 having deletion of the tofI/tofM/tofR cluster, which are defective in the LuxI/LuxR-type quorum-sensing (QS) system of B. glumae, for comparative analyses of the two major regulatory systems (the qsmR gene and the tofI/tofR QS). From this study, genes differentially expressed depending on the qsmR gene and those on the tofI/tofR QS were identified and characterized with 336gr-1 and 411gr-6 strains, so biological functions regulated commonly by both regulatory systems and specifically by each of them could be predicted. To determine regulatory functions of the qsmR gene and the tofI/tofR QS in extended strains of B. glumae, we generated the mutant derivatives that are defective in each of the regulatory systems with additional twelve strains of B. glumae isolated from rice fields in Louisiana. Most of the strains showed the pattern of 411gr-6, in which virulence function was retained after deletion of the tofI/tofR QS but abolished by the deletion of the qsmR gene. 2) Identify metabolic conditions and signals affecting the function ofqsmRand virulence Experimental conditions were established to determine the influences of different carbons sources on the function of the qsmR. The main works for this goal are planned to begin from the second project year. 3) Investigate the dynamics ofqsmRfunction associated with bacterial adaptation to changing environments The main work planned for this goal in the first year was to survey B. glumae strains isolated from Louisiana rice fields in terms of the frequency of naturally avirulent strains and characterize the functionality of the qsmR gene in those strains. Unfortunately, occurrence of bacterial panicle blight was very low this year, so only a few strains could be obtained this year and all of them were virulent. As an alternative strategy, we tested the naturally avirulent strains in our strain collection for their functionality of the qsmR gene. Interestingly, all the 11 avirulent strains tested, only 257sh-1 could restore its virulence through the in trans expression of the functional qsmR gene from the virulent strain 336gr-1, indicating that other factors than qsmR are also responsible for the natural avirulence trait.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
De Paula, T., J. Padilla, I. Barphagha, J. C. E. Ontoy, J. S. Bruno, and J. H. Ham. 2022. Characterization of qsmR functioning as a master regulator beyond the quorum-sensing system for the pathogenesis of Burkholderia glumae in rice. Phytopathology 112:S3.118.
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