Role of GRAS Transcription Factors in Tomato Disease Resistance and Basal Defense

The research problem: Bacterial spot and bacterial speck diseases of tomato are causedby strains of Xanthomonas campestris pv. vesicatoria (Xcv) and Pseudomonas syringae pv.tomato (Pst), respectively. These bacteria colonize aerial parts of the plant and causesignificant losses in tomato production worldwide. Protection against Xcv and Pst bycultural practices or chemical control has been unsuccessful and there are only limitedsources of genetic resistance to these pathogens. In previous research supported in part byBARD IS-3237-01, we extensively characterized changes in tomato gene expression uponthe onset of spot and speck disease resistance. A remarkable finding of these studies wasthe inducibility in tomato leaves by both Xcv and Pst strains of genes encodingtranscriptional activator of the GRAS family, which has not been previously linked todisease resistance. Goals: Central goals of this research were to investigate the role of GRAS genes in tomatoinnate immunity and to assess their potential use for disease control.Specific objectives were to: 1. Identify GRAS genes that are induced in tomato during thedefense response and analyze their role in disease resistance by loss-of-function experiments.2. Overexpress GRAS genes in tomato and characterize plants for possible broad-spectrumresistance. 3. Identify genes whose transcription is regulated by GRAS family. Our main achievements during this research program are in three major areas:1. Identification of tomato GRAS family members induced in defense responses andanalysis of their role in disease resistance. Genes encoding tomato GRAS family memberswere retrieved from databases and analyzed for their inducibility by Pst avirulent bacteria.Real-time RT-PCR analysis revealed that six SlGRAS transcripts are induced during theonset of disease resistance to Pst. Further expression analysis of two selected GRAS genesshowed that they accumulate in tomato plants in response to different avirulent bacteria orto the fungal elicitor EIX. In addition, eight SlGRAS genes, including the Pst-induciblefamily members, were induced by mechanical stress in part in a jasmonic acid-dependentmanner. Remarkably, SlGRAS6 gene was found to be required for tomato resistance to Pstin virus-induced gene silencing (VIGS) experiments.2. Molecular analysis of pathogen-induced GRAS transcriptional activators. In aheterologous yeast system, Pst-inducible GRAS genes were shown to have the ability toactivate transcription in agreement with their putative function of transcription factors. Inaddition, deletion analysis demonstrated that short sequences at the amino-terminus ofSlGRAS2, SlGRAS4 and SlGRAS6 are sufficient for transcriptional activation. Finally,defense-related SlGRAS proteins were found to localize to the cell nucleus. 3. Disease resistance and expression profiles of transgenic plants overexpressing SlGRASgenes. Transgenic plants overexpressing SlGRAS3 or SlGRAS6 were generated. Diseasesusceptibility tests revealed that these plants are not more resistant to Pst than wild-typeplants. Gene expression profiles of the overexpressing plants identified putative direct orindirect target genes regulated by SlGRAS3 and SlGRAS6.  Scientific and agricultural significance: Our research activities established a novel linkbetween the GRAS family of transcription factors, plant disease resistance and mechanicalstress response. SlGRAS6 was found to be required for disease resistance to Pstsuggesting that this and possibly other GRAS family members are involved in thetranscriptional reprogramming that takes place during the onset of disease resistance.Their nuclear localization and transcriptional activation ability support their proposed roleas transcription factors or co-activators. However, the potential of utilizing GRAS familymembers for the improvement of plant disease resistance in agriculture has yet to bedemonstrated.