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The Analysis of WRKY70/ LURP1 Dependent Defense Mechanism in Arabidopsis and Tomato



The Analysis of WRKY70/ LURP1 Dependent Defense Mechanism in Arabidopsis and Tomato


Ayesha Baig

Doctor of Philosophy, Graduate Program in Plant Biology

University of California, Riverside, March 2013

Dr. Thomas Eulgem, Chairperson

The Arabidopsis LURP1 gene of LURP gene cluster (Late Up-regulation in Response to Hpa) is a member of a 15-member gene family termed LOR (LURP-one related). Of all Arabidopsis LOR family members, only LURP1 shows an unusually pronounced up-regulation in response to Hyaloperonospora arabidopsidis (Hpa) where as LOR1 shows strong constitutive expression based on microarray data. Reverse genetic studies using transposon insertion mutants revealed an important role of LURP1 in disease resistance mediated by the R-gene RPP5, while LOR1 plays a significant role in basal defense against Hpa. The promoter swap expression lines -1004pLURR1::LOR1 and -1093pLOR1:: LURP1 were constructed to determine if the promoter or slight differences in their protein sequences are important for the differences in their defense-related roles. Confocal microscopy with stably expressed GFP fusion proteins showed GFP-LURP1 and GFP-LOR1 localized at the plasma membrane and in to the nucleus. Homozygous GFP-LURP1 and GFP-LOR1 restore wild type immunity in the respective mutant backgrounds during compatible and incompatible interaction with Hpa. Yeast two hybrid (Y2H) screens with LURP1 and LOR1 as baits identified no significant interacting partners from a screen of 2.3 x 105 cDNA clones.

In Arabidopsis LURP expression is controlled by the WRKY70 transcription factor. Multiple transgenic tomato lines (cultivar VFNT Cherry tomato) containing a Hpa-responsive GUS reporter gene containing a promoter fragment of the LURP member CaBP22 (pCaBP22-333::GUS) , responded to the defense inducing chemicals SA, BTH and DCA indicating that the mechanism of LURP regulation is likely conserved between Arabidopsis and tomato. Phylogenetic analysis revealed that WRKY70, which is a single copy gene in Arabidopsis, has two orthologs in tomato termed SlWRKY70a and SlWRKY70b. I made three silencing constructs to individually or co-silence SlWRKY70a and SlWRKY70b. TRV-VIGS silencing construct in homozygous tomato plants containing pCaBP22-333::GUS reporter gene showed that at least one of the two tomato WRKY70 orthologs is required for mediating responses to BTH or DCA defense induction. Thus, the WRKY70/LURP regulatory module is likely conserved between Arabidopsis and tomato.

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