Final report for CDFA contract number #06-0222

BIOLOGY OF THE XYLELLA FASTIDIOSA-VECTOR INTERFACE

Project Leader:	Researcher:

Rodrigo Almeida	Nabil Killiny

rodrigoalmeida@berkeley.edu	nkilliny@berkeley.edu

Dept. Environmental Science, Policy and Management

University of California, Berkeley, CA 94720

Reporting period:

The results reported here are based on work conducted from November 2009 to February 2010. This project is a continuation of project 06-0222, awarded in 2006.

List of objectives, and description of activities conducted to accomplish each objective

The broader goal of this project is to develop a strategy to block vector transmission of Xylella fastidiosa by disrupting insect-pathogen interactions. Our research has used approaches that build on each other to i) generate information on the interactions in this system and ii) test ideas on how to block transmission. First, we need to understand how X. fastidiosa colonizes sharpshooters, more specifically, how it interacts with the chitinous surface of the foregut of vectors. Although there is still much to be studied about this system, we now have a hypothesis for X. fastidiosa colonization of vectors, from a cellular and molecular perspective, that was proposed based on molecular, biochemical and biological data (Killiny and Almeida 2009a,b). In this hypothesis, cell initial adhesion (A) to the cuticle of sharpshooters is mediated by afimbrial adhesins. These proteins have affinity for N-acetylglucosamine, which can be used to block this interaction in vitro. Although we are continuing research on all the steps of colonization present in our hypothesis, we believe that step A is a good target for disruption because biofilms have still not been formed within insects.

In the laboratory we were able to block X. fastidiosa adhesion to leafhopper foregut extracts by saturating solutions with N-acetylglucosamine, its dimer and trimer. Essentially, cells bound to these carbohydrates in solution rather than to the foregut extracts. However, to test this approach with transmission experiments we needed to develop an artificial diet system to deliver cells in a transmissible state to leafhoppers, so that normal adhesion to vectors would occur and similar competition tests performed. We modified a medium for cell growth developed earlier (Almeida et al. 2004) and found that X. fastidiosa grown in this new environment was hyperadhesive and transmissible by vectors (Killiny and Almeida 2009b). In fact, transmission rates were close to 100% for one medium (XFM-pectin), which is interesting when compared to the commonly used medium PWG, for which no transmission was observed. A byproduct of this effort was that X. fastidiosa’s gene expression profile was dramatically different from cells grown in traditionally used media, and that is now being explored by the research community in a varied of manners.

We now have i) some information on how X. fastidiosa interacts with insect vectors, and ii) a protocol to deliver vectors ‘transmissible’ cells and, importantly, other compounds of interest that may block transmission. We used this approach to test if two groups of ‘generic’ molecules reduced X. fastidiosa transmission. In these experiments, cells and delivered to vectors through a diet system (figure on right) together with candidate transmission-blocking agents. After that insects are transferred to healthy grape plants for a pathogen inoculation access period. First, we tested if lectins, which bind to carbohydrates, reduced transmission by out-competing X. fastidiosa for adhesion sites in the foregut of vectors. We showed that two lectins that have affinity for N-acetylglucosamine and similar carbohydrates reduced transmission rates by half compared to the control. We also tested if transmission could be blocked by saturating adhesins on the surface of X. fastidiosa with molecules they have affinity for, like N-acetylglucosamine, which would lower the frequency of cell adhesion to vectors as the proteins responsible for that activity would be already bound to other molecules. Below are the experimental results of these tests, together with hypothetical models describing the mechanisms proposed to explain our data.

We are also using this artificial diet system to test the role of different X. fastidiosa mutants on vector colonization. Although our work has provided enough information to initiate trials that have been successful in reducing pathogen transmission, we believe that a better understanding of X. fastidiosa colonization of vectors will permit the identification of new targets for disruption of these interactions. Below is a list of different mutants we have tested, are testing, or hope to test in the near future, and their proposed role in vector transmission.

List of genes potentially implicated in the transmission and biofilm formation in vectors that are to be tested. We also hope to identify representative mutants for each category and test those by microscopy.

Gene	Gene product	Hypothetical role in transmission	Test status
hxfA1	Afimbrial adhesins	Initial attachment	Completed
hxfB1
Initial attachment	Completed
fimA2	Type I pilus	Polar attachment	Completed
pilB2	Type IV pilus	No role	Completed
fimA/pilB2	Type I/IV pili
Completed
pglA1	Pectinase	Gene regulation cascade	Completed
gumD3	Exopolysaccharides	Biofilm structure and protection	Completed
gumH3
Completed
rpfF4

Cell-cell signaling system

Gene regulation	Completed
rpfC4
Completed
rpfF/rpfC4
Completed
rpfB4
Ongoing
cgsA4
Ongoing
eal4
in 2010
eal/rpfF4
in 2010
tolC5	Secretion systems	Secreted proteins may be associated with transmission	in 2010
tps1
Ongoing
PD02186

AT-1 serine proteases

To be determined	in 2010
PD3136
in 2010
PD09506
in 2010
PD0218/PD09506
in 2010
PD0218/PD03136
in 2010
PD313/PD09506
in 2010
PD0218/PD0313/PD09506
in 2010
PD05286

Membrane proteins
Ongoing
PD13796
Ongoing
PD0528/PD13796
Ongoing
in vitro Evolution strains7	---------------------------
in 2010
1: Kirkpatrick, UC-Davis 2: Hoch and Burr, Cornell University 3: Cooksey, UC-Riverside 4: Lindow, UC-Berkeley 5: Gabriel, University of Florida 6: Igo, UC-Davis 7: Our lab

Results for some of the genes tested are shown below.

In summary, we now have a protocol to test X. fastidiosa transmission blocking agents. Furthermore, data we obtained allowed the development of a hypothesis for X. fastidiosa-sharpshooter interactions, and using this paradigm we tested candidate molecules to disrupt transmission. Our results show that this is a promising approach to limit the spread of Pierce’s disease that is system-specific.

Intellectual property issues you are aware of – none.

Peer-reviewed publications resulting from the work

N. Killiny and R. P.P. Almeida. 2009a. Xylella fastidiosa afimbrial adhesins are associated with cell attachment to leafhopper vectors and transmission to plants. Applied and Environmental Microbiology 75:521-8

N. Killiny and R. P.P. Almeida. 2009b. Host structural polysaccharide induces vector transmission of a bacterial plant pathogen. Proceedings of the National Academy of Science 106: 22416-20.

Describe how this work and its results help contribute to solving the PD problem in California

This work addresses half of X. fastidiosa’s life cycle, which occurs within sharpshooter vectors, and is responsible for pathogen spread between plants. Our goals are to generate information on how X. fastidiosa colonizes its vectors to develop approaches to block its transmission between plants. The experimental results obtained show that it is possible to significantly reduce X. fastidiosa transmission with ‘generic’ molecules that compete with cells for binding sites in vectors, or that bind directly to adhesins on the cell surface. In addition, the protocol used to induce a ‘vector transmissible state’ in X. fastidiosa resulted dramatic changes in gene expression of this pathogen, which apparently mimic its profile while colonizing plants. This finding is now being widely used by the X. fastidiosa community for a variety of tests, from gene regulation studies to tests of enzymatic activity.

Transmission rate of fimbrial and afimbrial adhesins, and DSF production mutants; two inoculation access periods were used (n=20/IAP).

Nabil

2010-03-16

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