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INVESTIGATOR: Kung, S.

PERFORMING INSTITUTION:
UNIVERSITY OF CALIFORNIA, BERKELEY
BERKELEY, CALIFORNIA 94720

HOMOLOGOUS RECOMBINATION AND THE EMERGENCE OF NOVEL PATHOGENS

NON-TECHNICAL SUMMARY: Emerging infectious plant diseases continue to affect agricultural production in both the United States and abroad. However, little research has been conducted on the evolutionary forces driving pathogen diversification and emergence. Xylella fastidiosa is a vector-borne xylem-limited bacterium that causes disease of economic importance in several crops such as grape, almond, and peach. The number of new diseases caused by X. fastidiosa has increased dramatically in recent years; however, the factors driving the emergence of these diseases and the mechanisms that allow the pathogen to adapt to new host environments are not understood. We recently found that X. fastidiosa is naturally competent and can recombine DNA acquired from the environment into its genome. We propose to characterize the biology of competency and determine what factors control its induction. We will investigate target genes identified by BLAST searches and previous microarray work for their role in transformation and recombination through molecular and biological assays. In addition, we will study the role of recombination in the evolution of X. fastidiosa's pathogenicity and determine what role if any its insect vector plays in promoting recombination events. We hypothesize that homologous recombination in X. fastidiosa occurs at high rates and that a constant reshuffle of genes throughout the genome results in the emergence of pathogenicity and new strains, which may drive epidemics. Understanding the role of horizontal gene transfer in the emergence of new diseases may help prevent future crop failure and famine resulting from pathogenic infections.

OBJECTIVES: Xylella fastidiosa is an economically important plant pathogen in the US with a wide host range. It causes disease in several agriculturally relevant crops such as grape, almond, peach and plum, in addition to trees and ornamental plants. The introduction of a strain causing disease in citrus is a major threat to the US industry. Although some X. fastidiosa diseases have been known for decades, many have emerged recently. Notably, several new diseases have been reported in California after the introduction of a generalist insect vector. We hypothesize that this polyphagous insect is responsible for transporting different strains of the pathogen into new hosts, increasing the frequency of recombination events and the emergence of new pathogens. This hypothesis is supported by both a dramatic increase in emerging X. fastidiosa diseases in California and a growing number of studies demonstrating that homologous recombination is a major contributor to this bacterium's genome plasticity. The role of homologous recombination on pathogen evolution has only recently been appreciated, primarily in the last decade after multi-locus sequence typing approaches have shown that recombination rates in many bacteria are much higher than mutation rates. However, experimental tests of the role of homologous recombination on pathogen adaptation, host range, virulence and other relevant processes have been extremely limited. We propose to use X. fastidiosa as a model system to ask such questions because it is naturally competent, has a wide host range, and virulence seems to be a complex trait derived from factors associated with pathogen multiplication and movement within the host's xylem vessel network. Understanding the biology and evolutionary role of homologous recombination in X. fastidiosa may lead to insights into why this pathogen has such a wide host range and new diseases have emerged after the introduction of a new vector into California. In addition, it will provide evidence to the role of this process in plant pathogen evolution and adaptation. Lastly, understanding the biology of competency in detail may generate useful information for research on other plant pathogenic bacteria. As bacterial plant pathogens are a major contributing factor to crop loss in both the United States and abroad, understanding the potential mechanisms through which new diseases emerge could play a vital role in mitigating crop losses, keeping American agriculture competitive and helping to end world hunger. We propose to address the following hypotheses: i) Low nutrient conditions and low cell densities induce natural competence and recombination. ii) Insect vectors provide ideal conditions for genetic exchange to occur between pathogen strains. iii) Homologous recombination events facilitate the adaptation of pathogens to new environments. Outputs include presentation of research at scientific meetings and grower groups impacted by X. fastidiosa diseases, publication of peer-reviewed research manuscripts, and sharing of data with research collaborators and other scientists as requested.

APPROACH: Objective 1. Characterization of natural competency and recombination in X. fastidiosa. Our first objective is to describe the biology of competency and recombination in X. fastidiosa through a series of bioassays coupled with gene expression microarrays. Because we are using sources of DNA that do not replicate within X. fastidiosa, our transformation rates include both DNA uptake and chromosomal integration. We expect that by coupling these approaches we will gain insights into its mechanisms, and, concurrently, identify target genes for future work. Objective 2. Recombination between strains in the foregut of X. fastidiosa's sharpshooter vector In addition to determing if recombination can affect host range and pathogenicity, we will determine what role if any the vectors of X. fastidiosa play in facilitating recombination events. We hypothesize that recombination between strains likely occurs in the foregut of the vectors, as the foregut represents a space-constrained low-nutrient environment where different strains are most likely to be exposed to one another. General protocols for insect transmission experiments, including rearing conditions, plants, etc will follow previous work done by our group. Sharpshooters will acquire X. fastidiosa through sachet, and fed an artificial diet medium containing an antibiotic resistance plasmid. We will then attempt to culture X. fastidiosa cells from the sharpshooter heads, and plate the suspensions on PWG with gentamicin and dilution plated on PWG to determine total cell count inside the foregut two days after insects feed on artificial diet system (following methods described in Almeida and Purcell 2003b). Objective 3. Recovery of pathogenicity in avirulent X. fastidiosa and adaptation to new host plants through recombination. To test for the recovery of pathogenicity through recombination, we will attempt to restore the virulence of both a single-gene mutant that results in a non-pathogenic phenotype and a naturally avirulent isolate through recombination with DNA from virulent strains. Replacing the tolC gene with a kanamycin resistance marker has been shown to result in a total loss of pathogenicity in grapevines, and no colonies were recovered from plants inoculated with tolC- (Reddy et al. 2007). The tolC- mutant grown with DNA, the mutant grown without DNA, wild-type X. fastidiosa and buffer will then be individually inoculated into 100 healthy grapevines each and grown in a controlled greenhouse environment for approximately 2 months. We will attempt to culture X. fastidiosa from leaf petioles near the inoculation sites to determine infection rate of the "rescued" mutants versus wild type. We do not expect to recover any colonies from plants inoculated with the tolC- mutant grown without DNA. We expect to recover colonies from a significant percentage of grapevines inoculated with mutants grown with the wild type copy of tolC, as only a single recombination event is required to recover pathogenicity and there will be a strong selection for recombinants in plants.

PROGRESS: 2011/08 TO 2013/07
Target Audience: Target audiences include the general scientific community, which is reached by publication of results in peer-reviewed journals and presentations at conferences and other venues. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This fellowship supported the PD in completing her Ph.D. In addition, we were able to train and mentor 3 undergraduate students under this project. The PD also gained valuable experience presenting scientific work, both by giving presentations at scientific meetings and preparing manuscripts for publication. How have the results been disseminated to communities of interest? Two manuscripts in peer-reviewed journals were published during the course of this project, and a third is currently under review for publication. In addition, newly generated knowledge was disseminated at scientific meetings, conferences, and other venues. We have also established collaborations with other research groups within the university and beyond and shared our research advances. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2011/08 TO 2013/07
What was accomplished under these goals? Financial support from this fellowship allowed us to focus on research activities and mentoring students. Support from the fellowship allowed the PD to complete her dissertation work and graduate from the Ph.D. program. We found several ways to increase the efficiency of recombination of naturally transformed DNA, and made several insights into how genetics, physiology, and biology affect this process. These results will not only facilitate genetic manipulation of this organism in the future, but also provides insight into the role of natural competence and recombination in the evolution and emergence of new pathogens.

PUBLICATIONS (not previously reported): 2011/08 TO 2013/07
1. Type: Journal Articles Status: Published Year Published: 2011 Citation: Kung, SH and Almeida RPP. 2011. Natural Competence and Recombination in the Plant Pathogen Xylella fastidiosa. Appl. Environ. Microbiol. 77:5278-5284.
2. Type: Journal Articles Status: Published Year Published: 2013 Citation: Kung SH, Retchless AC, Kwan JY, and Almeida RPP. 2013. Effects of DNA size on transformation and recombination efficiencies in Xylella fastidiosa. Appl. Environ. Microbiol. 79:1712-1717.
3. Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Kung, SH and Almeida RPP. Biological and genetic factors regulating competence in the bacterial plant pathogen Xylella fastidiosa.

PROGRESS: 2011/08/01 TO 2012/07/31
OUTPUTS: Over the past year, this fellowship has allowed us to mentor and train three undergraduate research assistants for our project. These students have gained experience with experimental design, analyzing data, and have learned a variety of laboratory techniques. In addition, results from our project have been disseminated in a variety of ways. The graduate student supported by this fellowship gave an oral presentation at UC Berkeley's annual Microbiology Student Group Symposium, and lab members have used data from this project in a variety of presentation forms to a wide range of audiences. Data was also presented at the annual investigator's meeting for AFRI NIFA fellows. The principal investigator of the lab also disseminated newly generated knowledge in research talks at conferences and other venues. We have also shared information with other researches working with Xylella fastidiosa, and have helped design a project for an undergraduate student at another university working under the supervision of one of our collaborators. PARTICIPANTS: Stephanie Kung is the graduate student supported by this fellowship. Rodrigo Almeida is her advisor. The fellowship provided financial support for Kung to continue her efforts towards her Ph.D. and provided her the opportunity to present her work and network with other fellowship recipients at the annual investigator's meeting. TARGET AUDIENCES: Target audiences include the general scientific community, which is reached by publication of results in peer-reviewed journals. In addition, Kung was able to mentor three undergraduate students over the past year and train them in scientific research principles and laboratory techniques. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

IMPACT: 2011/08/01 TO 2012/07/31
Financial support from this fellowship allowed us to spend the last year focusing on research activities and mentoring students. We currently have two articles for publication in peer-reviewed journals in preparation. We have found ways to increase the recombination efficiency of DNA acquired through natural transformation, which, along with its basic science implications, will make it easier for researchers to genetically manipulate X. fastidiosa for a variety of purposes. Recombination appears to be most efficient when there is at least 1kb of homologous flanking region present, and recombination efficiency decreases exponentially with the length of the non-homologous insert. In addition, we have identified several genes and other biological factors that seem to play a role in the natural transformation process.

PUBLICATIONS: 2011/08/01 TO 2012/07/31
Kung SH, Almeida RPP. 2011. Natural Competence and Recombination in the Plant Pathogen Xylella fastidiosa. Appl. Environ. Microbiol. 77:5278-5284.