Intellectual Merit. Gene flow between species is a natural process. The ubiquitous soil bacteria Agrobacterium tumefaciens and A. rhizogenes naturally transfer some of their DNA to plants, leading to production of crown gall and hairy root diseases. This ability to transfer DNA has been exploited for genetic engineering and is now the preferred method for delivering genes to plant cells. Despite the widespread use, many mechanistic questions remain about how the gene transfer takes place. In A. tumefaciens, the VirE2 protein plays a major role by coating the transferred DNA and facilitating its uptake into the nucleus of the plant cell. However, this does not appear to be a universal mechanism, as some strains of A. rhizogenes transfer DNA efficiently, but lack VirE2. Instead, these strains contain another protein, GALLS, that can substitute for VirE2, thus providing an alternate route for nuclear uptake of transferred DNA. Apparently, upon entry into plant cells, GALLS localizes inside the nucleus where it interacts with a bacterial pilot protein attached to the transferred DNA and with a plant protein (GALLS-Interacting Protein; GIP). GALLS is hypothesized to act as a "molecular tractor" to pull transferred DNA into the nucleus. To test this idea, multiple experimental approaches will be used to understand how GALLS and GIP promote gene transfer via a VirE2-independent pathway.
Broader Impacts. These studies will increase understanding of Agrobacterium-mediated gene transfer and identify additional unknown components of this extremely important gene delivery system. This project will promote education and training of a doctoral student at Oregon State University and a postdoctoral associate at Purdue University. In addition, minority undergraduates from Brooklyn College will be recruited to participate in summer research internships at Purdue. After their summer experiences, the students will continue to engage in related hands-on research at their home institution.
Plant genetic transformation (the introduction of new genes into plants) is a key process in agricultural biotechnology. Agrobacterium-mediated transformation is the most widely used method for genetically transforming plants. Agrobacterium species transfer Virulence effector proteins to plants, one of which (VirD2) is attached to T- (transferred) DNA. Thus, Vir protein transfer results in T-DNA transfer. Other virulence effector proteins also aid in transferring T-DNA to plant cells, targeting it to the nucleus, and integrating it into the plant genome. One of these proteins, VirE2, plays a key role by protecting single-strand T-DNA in the plant cell and perhaps by helping direct it to the nucleus. Agrobacterium tumefaciens strains mutant in virE2 are almost completely avirulent. Interestingly, some A. rhizogenes strains lack a virE2 gene yet are completely virulent. These strains encode another protein, GALLS, that can substitute for VirE2 protein, despite the fact that these two proteins differ substantially in amino acid sequence and encoded protein domains. The purpose of this project was to investigate the mechanism by which GALLS helps effect transformation. We reasoned that, as an effector protein that enters the plant cells, GALLS probably interacts with plant host proteins. Using several techniques, we showed that GALLS interacts with all tested members of the Arabidopsis LSH protein family, which are putative transcription factors. Overexpression of many, but not all, LSH family members in Arabidopsis substantially enhanced (~10-fold) transformation susceptibility to both A. tumefaciens and A. rhizogenes. Most surprisingly, overexpression of two LSH family members allowed plants to be transformed by Agrobacterium strains lacking both virE2 and GALLS. Such VirE2- and GALLS-independent transformation had never previously been shown. Because LSH proteins are putative transcription factors, we investigated whether their overexpression would alter plant transcription patterns. Indeed, RNA-seq analysis indicated that ~300 genes showed altered transcript abundance in LSH overexpressing plants versus wild-type plants. Among those genes that showed increased expression were two WRKY transcription factors which are negative regulators of plant defense responses. Increasing their expression would down-regulate plant defenses, which may increase transformation susceptibility. Indeed, we showed that overexpression of one of these WRKY transcription factors increased susceptibility to Agrobacterium-mediated transformation. Because Agrobacterium-mediated transformation plays such a key role in agricultural biotechnology, and because many agronomically important crop species are recalcitrant to transformation, any mechanism to increase plant susceptibility to transformation would greatly accelerate development of transgenic crops that show improved agronomic characteristics. In addition to training young scientists and minority students who visited the laboratory in the summer, this project has opened up new avenues for improving plant transformation.
