Agrobacterium species transform plant cells by targeting a portion of plasmid-encoded bacterial DNA to the host nucleus. Genetic transformation by A. tumefaciens and A. rhizogenes requires secreted effector proteins. The tumor-inducing (Ti) plasmid from virulent A. tumefaciens encodes VirE2, a secreted single-stranded DNA-binding protein required for efficient transformation of hosts. Some strains of A. rhizogenes harbor root-inducing (Ri) plasmids that lack the virE2 gene, and contain the GALLS gene instead. GALLS can fully substitute for the lack of virE2. The GALLS gene encodes two independently translated proteins: full-length (GALLS-FL) and a more abundant C-terminal domain (GALLS-CT) protein (collectively called GALLS). Both proteins are secreted from the bacterium into plant cells during transformation. GALLS-FL is a putative strand-transferase that is essential for GALLS-mediated transformation of all hosts. GALLS-CT is not required for transformation, but can significantly stimulate gene transfer to many plant species, including Arabidopsis thaliana.
Host responses to Agrobacterium effectors are not well-understood. VirE2 interacts with host proteins in planta, however the exact roles and importance of these host proteins during transformation remain unclear. Host responses to GALLS-FL and GALLS-CT effectors have not been investigated before. We hypothesized that GALLS interacts with A. thaliana proteins that are important for Agrobacterium-mediated transformation. We also hypothesized that GALLS-CT, which is important for transformation of some hosts, may interact with host-specific proteins to alter host gene expression in favor of Agrobacterium infection.
Using yeast two-hybrid screens, we identified LHS10, a putative host transcription factor that binds both GALLS proteins in planta. Overexpression of LSH10 in transgenic A. thaliana stimulated both GALLS- and VirE2-mediated transformation (transient and stable). RNA-Seq analysis of hypersusceptible plant lines overexpressing LSH10 revealed differential expression of many defense related genes, including up-regulation of WRKY38. WRKY38 is a transcription factor that suppresses expression of plant defense genes. Expression of WRKY38 from the CaMV 35S constitutive promoter in transgenic A. thaliana increased host susceptibility to Agrobacterium-mediated transformation. LSH10 may stimulate Agrobacterium-mediated transformation by lowering plant defense responses, leading to improved transformation efficiency.
To identify the effects of GALLS-CT on host responses, we created transgenic A. thaliana that expressed GALLS-CT from an inducible promoter. GALLS-CT expressed in planta for six hours prior to Agrobacterium infection stimulated transient transformation. RNA-Seq analysis of transgenic plants producing GALLS-CT showed down regulation of several genes encoding heat shock and small heat shock molecular chaperones important for protein processing. Several of these heat shock proteins are involved in stabilizing plant disease resistance (R) proteins. Down regulation of molecular chaperone genes may lead to destabilization of defense response proteins, which may dampen host defense responses and improve Agrobacterium-mediated transformation frequencies in A. thaliana.
Despite the importance of Agrobacterium-mediated transformation in agriculture, biotechnology, and plant science, plant responses to Agrobacterium are not well-understood. Genetic transformation of plants by Agrobacterium tumefaciens and A. rhizogenes requires secreted effector proteins VirE2 or GALLS. No studies prior to our work have investigated whether any host proteins interact with GALLS-FL or GALLS-CT, or how GALLS-CT may alter host-specific responses to stimulate transformation. We discovered that GALLS interacts with host protein LSH10. Overexpression of LSH10 made plants hypersusceptible to GALLS- and VirE2-mediated transformation, possibly by dampening host defense responses via up-regulation of WRKY38. We also observed that GALLS-CT produced in transgenic A. thaliana stimulated GALLS-mediated transient transformation. Transgenic plants that produced GALLS-CT showed decreased expression of genes encoding heat shock molecular chaperones involved in stabilizing disease resistance proteins. Our studies suggest that LSH10 and GALLS-CT may stimulate transformation by dampening host defense responses against Agrobacterium. These results shed light on a potentially new defense response pathway in A. thaliana, as well as how bacterial effector proteins may alter host responses to promote infection. Our findings may also prove useful in improving transformation frequencies in recalcitrant hosts.