Source: OREGON STATE UNIVERSITY submitted to
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Feb 15, 2008
Project End Date
Feb 14, 2013
Grant Year
Project Director
Chang, J. H.
Recipient Organization
Performing Department
Non Technical Summary
Rhizobia are important bacteria that form beneficial relations with legumes such as soybean. Rhizobia are one of the few bacteria that can convert atmospheric nitrogen into biologically usable forms. This is a critical process because nitrogen is an essential component of many biological building blocks yet is often the most limiting nutrient in soil. Consequently, agricultural practices often include applying commercially produced fertilizers or by using legumes and Rhizobia through crop rotation and green manures. However, nitrogen fixation can only occur in host-specific association with legumes. Recently, it was discovered that many strains of Rhizobia share a host-association mechanism commonly found in pathogens of humans and plants. This mechanism functions like a molecular syringe to inject molecules, called type III effectors, from the bacterium directly into the host cells. Type III effectors benefit the bacterium by reprogramming the host cell to block host immunity and facilitate infection. Ironically, in the case of plant pathogens, type III effectors can sometimes betray the pathogen to the host by eliciting host immune responses. Therefore, type III effectors are host-specific determinants of pathogens and we hypothesize that they have similar functions in Rhizobia. Our goal is to identify the type III effectors from several strains of Rhizobia and test for their effects on host-specific nodulation. Understanding the functions of type III effectors has potential towards altering and enhancing host ranges of Rhizobia.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Goals / Objectives
(1) Identify the collections of type III effectors from four strains of Sinorhizobium fredii and four strains of Bradyrhizobium japonicum. (2) Characterize the type III effectors from one strain of S. fredii for effects on nodulation and soybean compatibility.
Project Methods
We have developed a two-step, high throughput functional screen to identify type III effectors of Rhizobia. In the first step, we use a fluorescence activated cell sorter (FACS) to screen and clone genes that are regulated by ttsI. The ttsI protein regulates the expression of the type III secretion system (TTSS) and the type III effectors. The TTSS forms the secretion apparatus that injects type III effectors directly into host cells. In the second step, we use TTSS-dependent delivery of proteins to identify which encode type III effectors. This is an important step because ttsI regulates the expression of many genes and only a subset encodes type III effectors. We have already shown that our adaptation of the screen will work for identifying type III effectors of Rhizobia. To identify perceived type III effectors, or avirulence proteins of Rhizobia, we will screen for loss of compatibility between S. fredii and normally compatible cultivars of soybean. We will introduce each of the type III effectors from an incompatible strain of S. fredii into a normally compatible strain. We will assay for reduction in nodulation efficiency relative to wild-type control strains.

Progress 02/15/08 to 02/14/13

Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Teaching and mentoring: The two first co-authors of the paper describing this work were PhD students supported from this award. Dr. Jeff Kimbrel is currently a postdoc at Lawrence Berkeley National Laboratory and Dr. William Thomas is currently a postdoc at OSU (with a different research group). Jason Cumbie was also supported from this award and will defend his PhD at the end of April 2013. Additionally, numerous undergraduate students received support from this award. Notably, two co-authors on the paper describing this work are now PhD candidate at Michigan State University (Caitlin Thireault) and Oregon State University (Allison Creason). Ryan Lilley and Stanley Lee are in medical and pharmacy school, respectively. How have the results been disseminated to communities of interest? Dissemination: Our lab participates in the Apprenticeships in Science and Engineering Program and have mentored two high school students. This work has also been presented as invited departmental talks at UC Riverside, U. Arizona, and Michigan State University. Finally, this work was published in PLoS Pathogens (see Publications). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

What was accomplished under these goals? Activities: We used next generation sequencing to generate draft genome sequences for members of Sinorhizobium fredii and Bradyrhizobium japonicum. A total of eight isolates from the two species were mined for type III effector genes, based on a coupled informatics-functional screen for type III function. In total, we functionally validated 47 type III effector families. This set of effector genes was consistent with candidate families previously reported and included nearly 30 more new families. Outcomes: In the context of host-pathogen interactions, type III effector genes are highly dynamic, indicative of a co-evolutionary arms race. In this work, we demonstrated that in the context of host-mutualism, type III effector genes are highly static. This was in spite of observations that the genomes are significantly more genetically diverse than pathogenic Pseudomonas syringae, our comparator species. Moreover, the type III effector genes of rhizobia were significantly more conserved in both presence/absence and sequence. Our analyses suggested that this extreme difference in conservation is best explained by differences in selective pressures of mutualism versus pathogenesis. We therefore concluded that the “mutualistic environment” model fits our data better than the more dogmatic “co-evolutionary arms” race model.


  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Mutualistic Co-evolution of Type III Effector Genes in Sinorhizobium fredii and Bradyrhizobium japonicum.

Progress 02/15/11 to 02/14/12

OUTPUTS: Activities: We have completed the screening of all type III effector candidate families for type III secretion system-dependent translocation from eight strains of Sinorhizobium fredii and Bradyrhizobium japonicum. The >200 type III effectors belong to 57 type III effector families. Our comparisons between S. fredii USDA207 and USDA257 shows that only three type III effectors are polymorphic between the two. Thus, there is an easy path for testing candidates for affecting nodulation restriction in an R-gene dependent manner. We also completed the mining of type III effectors from Mesorhizobium loti MAFF303099. This strain is interesting because, to date, it appears that MAFF303099 recently acquired its type III secretion system-encoding loci since other M. loti strain appear to lack homologous sequences. Thus, our analysis provides insights into how type III effector genes are born or acquired. Teaching and mentoring: Three graduate students are supported by this grant. One recently completed his PhD and another is scheduled to defend. Dissemination: I participated in an outreach program in the Apprenticeships in Science and Engineering Program and mentored one High School student for a 10-week period. I presented results from this project to a departmental audience at UC Riverside, Michigan State University and will also give a presentation at the University of Arizona next month. PARTICIPANTS: Jeff Chang (PI) Jeff Kimbrel Bill Thomas Jason Cumbie Collaborators: Joel Sachs (UCR) TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Outcome: We used the set of confirmed type III effectors to test the two competing paradigms that model co-evolutionary host-mutualist interactions. In the arm race, partners are predicted to exhibit genetic patterns indicative of diversification. In the competing paradigm, the two partners are predicted to exhibit genetic patterns indicative of stasis. Our analysis of type III effector families show that, in contrast to those of phytopathogens, collections exhibit very little diversity in content. Within families, we found very little evidence for diversifying selection, very little association to regions with evidence for horizontal gene transfer, and few cases of pseudogenization. We therefore concluded that the "mutualistic environment" paradigm models the evolution of type III effectors of rhizobia.


  • No publications reported this period

Progress 02/15/10 to 02/14/11

OUTPUTS: Activities: In our last report, we had completed the computational mining for candidate type III effector genes from eight rhizobial strains (three species). We leveraged finished sequences from Rhizobium NGR234 and B. japonicum USDA110 to order contigs. All genomes have been annotated and we have completed a "comparative genomics" study of the genomes. This past year, we also sequenced the genome for B. japonicum USDA122 and mined it for its candidate type III effector genes. At this point in time, we have completed the cloning of 217 candidate genes from the nine strains. These 217 belong to 125 candidate effector families. There are more candidate genes because we decided to test multiple family members if they had sufficient allelic variation. All candidate genes have been fused to our reporter for type III secretion system dependent (T3SS) delivery and all gene fusions have been mobilized into P. syringae pv tomato DC3000. We are currently assaying candidates for T3SS-dependent delivery. Of the 140 candidate type III effectors from the S. fredii and M. loti strains, 33 have been tested in triplicate. Four have been confidently classified as encoding type III effectors and eleven have strong evidence for encoding type III effectors. More than 40 of the 77 candidate genes from B. rhizobium strains have been tested in triplicate for T3SS-dependent delivery. Nine genes have been confidently classified as encoding type III effectors. Another nine have strong evidence for encoding type III effectors. The remaining lack sufficient evidence for such classification. Teaching and mentoring: I continue to mentor the same three graduate students supported by this grant. Dissemination: I participated in an outreach program to the Academy for Lifelong Learning. My target audience was mature adults. I helped advance their knowledge in genomics and genomic technology. I presented this project to a departmental audience at the University of Toronto. PARTICIPANTS: Jeff Chang (PI) Jeff Kimbrel (graduate student; GS) Bill Thomas (GS) Jason Cumbie (GS) Rebecca Pankow (UG) Andres Alverez (UG) Stanley Lee (UG) Hari Krisnan Wolfgang Streit This research project provides graduate and undergraduate students training in genomics, computational biology, high throughput genomics, and host-microbe interactions. TARGET AUDIENCES: Efforts: I participated in a class for the Academy of Lifelong Learning, eligible for all mature adults. I gave a 40-minute long lecture on genomics and its impacts to society. I covered contributions to molecular medicine, food security, bioenergy, as well as anthropology and human evolution. I, along with several others gave participants a tour of the Center for Genome Research and Biocomputing. Participants were shown the capillary as well as Illumina next generation sequencing machines, confocal microscope and computational cluster. Overall, the class was well received and we received positive feedback. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

In our previous report, we indicated that a preliminary conclusion is that type III effectors are fairly conserved within and between rhizobial species. As we have working towards refining our list of type III effectors, this conclusion still hold true and is actually more supported now that most of the less-conserved candidates appear not to encode type III effectors. It also appears that rhizobia encode fewer type III effectors than pathogenic P. syringae. Change in knowledge: trainee, graduate student Jeff Kimbrel has acquired skills in computational biology and has authored or co-authored three manuscripts.


  • No publications reported this period

Progress 02/15/09 to 02/14/10

OUTPUTS: We have completed the mining of genome sequences for candidate type III effector genes from Rhizobia NGR234 (considered a S. fredii), S. fredii USDA207 and 257, M loti MAFF303099, B. japonicum USDA110, 123, and 124. We have completed the cloning of candidate type III effector genes from the first four strains and are more than 50% complete with the remaining strains. Of the 74 S. fredii candidates (all isolates), we have tested 37 and 13 were delivered. We have cloned 24 candidates from M. loti but none have been tested. We found a much higher number of candidates in B. japonicum isolates. This is not surprising considering its genome size is almost twice as large as the genome of S. fredii. Of the 236 candidates from all isolates of B. japoncium, we have cloned 110. Eleven have been tested and 7 were delivered. Replicate delivery assays are currently ongoing thus, what we currently classify as confirmed type III effectors should be viewed as a tentative classification. Teaching and mentoring: These activities supported three graduate students and four undergraduate students. We have also used these activities to mentor one undergraduate summer student from Heritage University, an institution for students with American-Indian heritage. This year, we are mentoring an undergraduate summer student from Reed College, an undergraduate teaching institution. Events: the PI organized and led a Genomics, Bioinformatics, and Systems Biology Symposium and Workshop, a two-day event at OSU. PARTICIPANTS: Jeff Chang (PI) Jeff Kimbrel (graduate student; GS) Bill Thomas (GS) Jason Cumbie (GS) Cait Thireault (undergrad student UG) Rebecca Pankow (UG) Ryan Lilley (UG) Allison Smith (UG) Andres Alvarez (UG) Training: Jayme Stout (UG; Heritage) Gleb Bazilevsky (Reed College). TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Findings are expected to impact the understanding of the role of type III effectors in the context of mutualism.


  • W. J. Thomas, C. A. Thireault, J. A. Kimbrel, and J. H. Chang (2009). Recombineering and stable integration of the Pseudomonas syringae pv syringae 61 hrp/hrc cluster into the genome of the soil bacterium Pseudomonas fluorescens Pf0-1. Plant J. 60(5): p. 919-28.
  • J. S. Cumbie, R. C. Pankow, W. J. Thomas, and J. H. Chang (2010) AutoSPOTs: Automated Image Analysis for Enumerating Callose Deposition in 10th Japan-US Seminar: Genome-Enabled Integration of Research in Plant Pathogen Systems, K. Akimitsu, et al., Editors. 2010, APS press: St. Paul, MN. Invited book chapter.

Progress 02/15/08 to 02/14/09

OUTPUTS: We have made measurable progress towards meeting the goals of our proposal. These are outlined in points 1 and 2. We have also made considerable progress in developing tools for downstream characterization of Rhizobia type III effectors. These are outlined in points 3 and 4. 1) We completed the genome sequences for five strains of Rhizobia. We used paired-end sequencing with the Illumina 1G Genome Analyzer and the de novo assembler Velvet to assemble the short reads (table 1). We generated between 30 to 55 million reads. Largest contig sizes ranged from 300 kb to 640 kb. The N50 ranged from 5.5 kb to 95 kb and the N50 number ranged from 11 to 29. 2) We developed a hidden markov model (HMM) to identify tts-boxes and a script to score translated ORFs for putative type III secretion translocation signals. We identified as little as 11 to as many as 67 putative tts-boxes. We are currently cloning candidate type III effector genes by cloning both full-length (~100 bp upstream of the tts-box + ORF) and ORF only fragments from three reference strains. We just recently began testing candidates for delivery. Five out of eleven tested proteins from B. japonicum USDA110 were delivered and are considered type III effectors (we are testing a total of 48 ORFs). We are also close to testing 26 ORFs from NGR234 and 26 from MAFF303099. We would like to point out that others have reported similar approaches to mine candidate type III effector genes from completed genomes. Our work is distinctly different in that we have a functional assay to test for direct delivery into host cells. 3) We have developed a new system for type III effector delivery. We used recombineering and Tn5 to stably integrate the entire type III secretion system encoding cluster into the genome of non-host associated P. fluorescens Pf0-1. We have shown this strain expresses a functional type III secretion system and can be used for characterizing type III effector proteins for their in planta functions. This work is near completion and we will submit it to The Plant Journal within one month's time. 4) We have also developed a program called SPOTS that automates the enumeration of callose spots. There are two advancements in our program. First, with user-defined inputs, SPOTS will automatically enumerate and average callose spots from pictures derived from the same treatment. Secondly, we have included statistical analyses to compare between treatments and to identify possible outliers. We expect to complete this work soon and will submit to Molecular Plant-Microbe Interactions. PARTICIPANTS: PIs/PDs: Jeff Chang assisted with some of the wet-lab work. The PI also guides the individuals below and helped write the manuscripts. Graduate students: Jeff Kimbrel and William Thomas. Graduate students have been involved in mining genome sequences, cloning and testing candidate type III effector genes. Newly added participant: Graduate student Jason Cumbie who is involved in genome assembly and mining for type III effector genes. Undergraduate students: Cait Thireault and Ryan Lilley. Assist in cloning of candidate type III effector genes and optimizing nodulation assays. Training and professional development. Three graduate and two undergraduate students are currently being trained. TARGET AUDIENCES: The PI is a co-instructor for introductory biology and teaches nearly 1000 students each academic year. The PI has introduced a lecture on Rhizobia-legume interactions. The PI also actively recruits students from the class for research opportunities across Oregon State University. This past year, more than 25 students were helped by the PI. All five undergraduates currently in the PI's lab were recruited from the class. The PI is also a co-instructor for a graduate level genome organization, structure, and maintenance course. The PI recently revamped the course to include more lectures on prokaryotic genome structure and high-throughput sequencing methodology. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Change in knowledge: We generated much larger contigs than we had originally anticipated. To our surprise, our initial observation that ttsI functions heterologously in Pseudomonas syringae could not be generalized to all genes with tts-boxes. Both led to changes in actions. Change in actions: Because of the two aforementioned changes in knowledge, we are using a bioinformatic screen rather than the functional screen to identify candidate type III effectors of Rhizobia as originally proposed. Also, because of change in knowledge 1, we no longer need to carry out goal #2 of the proposal, which was to use a variety of labor-intensive methods to clone out full-length candidate genes.


  • No publications reported this period