Co-PIs: Wilhelm Gruissem (ETH Zurich, Switzerland), Adriana Bernal (Universidad de Los Andes, Colombia)
Cassava bacterial blight, incited by Xanthomonas axonopodis pv. manihotis (Xam) is a serious threat to cassava plants in several developing countries in both Africa and South America. This project will first focus on the genome characterization of Xam to sequence, annotate, and characterize the constellation of bacterial effector (virulence) proteins that are present in naturally occurring field isolates of this important bacterial pathogen. The ability to rapidly and inexpensively determine the genome sequence of natural field isolates of Xam will provide novel insights into the evolution of pathogen virulence and the allelic diversity of effector genes that occur in natural populations. The genome assemblies of Xam will be employed to identify new sources of disease resistance in wild populations of cassava plants. Previous work has demonstrated that the PthB effector is present in all strains of the pathogen and is essential for the pathogen to cause disease. Work in this project will engineer cassava plants to recognize the PthB protein to provide effective and durable disease resistance. Results from the project are expected to provide a durable source of resistance to this very important bacterial pathogen of cassava. In addition, the knowledge generated in this project will greatly benefit cassava researchers in both Africa and Latin America.
The project will have a major impact in cassava breeding and will hence benefit smallholder agriculture in developing countries from Latin America, Africa and Asia. The collaboration between experts in genomics, plant-microbe interactions and cassava transformation with scientists who work on this important crop in developing regions will have synergistic effects in the deployment of new resistant varieties to this and other diseases. Furthermore, two Colombian graduate students will be trained under this project. The training of Colombian scientists in cutting edge technology at the Staskawicz and Gruissem laboratories will greatly benefit Colombian agricultural science.
Access to project outcomes Genomic sequences of Xam will be made available at NCBI and at a dedicated project website that will be created for this project to both facilitate the sharing of results between the groups involved, and the prompt dissemination of reliable data for the research community. The project web site, which will be accessible from http://plantbio.berkeley.edu/~stask, will include a description of bioinformatic algorithms developed for the assembly and annotation of the genomes, information on the bacterial strains analyzed, and description of the constructs and cassava plants developed in the project. All of these resources will be made freely available to the research community.
Cassava is an important food crop in Africa, Asia, and South America. The goal of our project was to make progress toward the development of cassava varieties resistant to cassava's major bacterial pathogen, Xanthomonas axonopodis pv. manihotis (Xam). Plant disease resistance is achieved when a plant genome encodes a resistance (R) protein capable of recognizing a bacterial effector protein. Effectors are a functionally diverse group of proteins that the pathogen injects into plant cells to cause disease. To identify an cognate R genes that, once transformed into the plant, would confer durable resistance in the field, we first had to identify conserved effectors used by Xam strains to cause disease, as these would be the least likely to be lost or modified in order to evade recognition in the plant. We used high throughput sequencing technology to sequence the genomes of 65 diverse Xam strains and then predicted the effector repertoire of each strain using a computational pipeline which we have made available for use by other scientists. We identified a group of effectors that were present in all sequenced strains as the best candidates for which to identify a cognate R gene. Systematic mutational analysis was carried out to measure the virulence contributions of each conserved effector. We found that the effectors AvrBs2, XopZ, XopX, XopAOI, XopV, XopN, TAL14, and TAL20 contributed to virulence and/or symptom formation. We are currently developing an in vitro method for assaying resistance responses to these effectors in different cassava cultivars. A marker of a resistance reaction is localized cell death called a hypersensitive response (HR). Resistance responses (HR) were identified in Nicotiana benthamiana and N. tabacum in response to TALE1Xam and XopAOI. R genes from these species can be identified and tested for functionality in cassava. Transcription activator like effectors (TALEs) are bacterial effectors that are translocated into the plant nucleus where they bind DNA and directly activate plant genes that provide a favorable environment for the pathogen. It is possible to predict the DNA sequence that a TALE will bind based on its amino acid sequence. We proposed to engineer a gene promoter containing a Xam TALE binding site which we would put upstream of a resistance-triggering gene. The resulting construct would be induced by Xam TALEs during infection of transformed cassava. We identified two genes to trigger resistance: RXam2(D492V), a resistance gene from cassava mutated to be autoactive, and avrGf2, an effector from another xanthomonad that is recognized in cassava by an as of yet unidentified R gene. These genes were cloned downstream of a TALE-inducible promoter and transformed into cassava. No embryos were recovered for the avrGf2 constructs. This is probably because the construct was "leaky", meaning that some avrGf2 was expressed in the absence of the inducer, leading to uncontrolled cell death. However, we have 20 embryos growing after transformation with RXam2(D492V). These plants will be characterized in a few months. As another source of potential resistance, we turned to the known R genes EFR, Xa21, and Bs2 from other plant species that have been shown to be functional in heterologous hosts. We incorporated these genes into the cassava genome using Agrobacterium-mediated transformation and tested for increased resistance. Unfortunately, none of the confirmed transformants showed increased resistance to Xam. Therefore, we began screening natural cassava varieties for resistance and have identified cultivar KBH 2006/18 as showing increased levels of resistance to Xam. The molecular basis for this resistance is being elucidated. Much of this work was published in High-throughput genomic sequencing of cassava bacterial blight strains identifies conserved effectors to target for durable resistance (Bart et al. 2012). In summary, while we did not produce any fully resistant transgenic cassava plants, we did identify sources of possible resistance and ruled out the use of resistance genes previously shown to provide resistance in heterologous plant systems for developing cassava resistant to Xam. The data generated from this project will aid breeders in their attempts to develop disease tolerant/resistant cassava. This collaboration has brought together scientists from three continents to collectively tackle a major threat to food security. The training of "up and coming" young scientists has been a major benefit of this project. One student from Universidad de los Andes trained in the Staskawicz lab for two months in molecular tools for studying plant pathogenic bacteria and one student from Universidad Nacional de Colombia was trained in cassava transformation in the Gruissem lab. Talks and posters were presented at the 4th Xanthomonas Genomics Conference in Angers, France, the Global Cassava Partnership for the 21st Century (GCP21) conference in Kampala, Uganda, the International Conference in Plant Pathology in Beijing, China (ICPP 2013), the PSC-Syngenta Symposium in Stein, Switzerland, inter-departmental seminars at the ETH Zürich, the D-BIOL Symposium in Davos, Switzerland, and the Keystone Symposium on Plant Immunity in Big Sky, Montana.
