The MY1HS as a Method to Isolate TGA2.2 Partners

TGA2.2 is a transcription factor that mediates SA- and auxin- regulated gene expression. Being expressed constitutively, the activity of TGA2.2 must be regulated by either post-transcriptional modification or protein-protein interactions. Based on evidence for the later mechanism (Jupin and Chua, 1996; Stange et al., 1997), we tried to identify regulatory proteins that can interact with TGA2.2. A MY1HS strategy was established and employed using a TGA2.2 bait protein bound to an as-1 element found upstream of a HIS3 reporter gene, to screen a cDNA expression library from Arabidopsis and tobacco vegetative tissues for interacting proteins.

Recently, several different yeast hybrid screens were conducted in our group to isolate TGA interacting partners. For instance, Thurow (2002) was successful in isolating a tobacco NPR1 homolog using TGA2.1-GAL4BD as a bait in a classical Y2HS approach. In another approach, utilizing TGA2.1 in a split-protein sensor system that is based on the reconstitution of ubiquitin-mediated cleavage of a reporter protein, a DEAD box RNA-helicase and an E2 ligase were identified as TGA2.1-interacting partners (Krawczyk, 2003). Siemsen (2002) identified a scarecrow-like transcription factor that acts as a putative TGA2 coactivator protein using a MY1HS that utlized TGA2 from Arabidopsis as a bait protein and an Arabidopsis cDNA library, which was not fused to any activation domain.

In order to identify TGA2.2-interacting proteins, a MY1HS that utilizes TGA2.2 bound to the as-1 element was deployed (Figure 6.1). This novel screening method should identify prey proteins that are able to interact with TGA2.2 bait protein to assemble an as-1 bound activation complex, which will lead to the activation of a reporter gene. Similar approaches, termed as the one-and-a-half hybrid system (Serebriiskii et al., 2001), were described previously in a number of instances, such as

the identification of regulatory proteins that are involved in activation of ftz-dependent promoters (Yu et al., 1999).

The MY1HS screening conditions were optimized to work with established positive controls. Bait protein expression is considered to be a limiting criterion before performing any yeast hybrid screen (Toby and Golemis, 2001). The TGA2.2 bait protein was under the control of the Met25 promoter cassette that can regulate the transcription of the TGA2.2 coding sequence upon adding or omitting methionine in the culture medium, i.e., an "on" or "off" switch system for the expression of the TGA2.2 bait protein. TGA2.2 overexpression was toxic to the YRWH2 screening strain. The problem was resolved by lowering TGA2.2 expression levels in YRWH2 through methionine application to the screening media. The toxic effect of TGA2.2 on the YRWH2 cells might be due to TGA2.2 binding to other cis-elments found in yeast promoter regions. The quality of a cDNA library is critical to the success of any yeast hybrid screen. In the current study, the cDNA inserts (from the Arabidopsis and tobacco cDNA library plasmids) were driven by a truncated ADH1 promoter. The weak expression of cDNA inserts is considered as a serious pitfall for any yeast hybrid screen (Agatep et al., 1998). The detection of protein expressed from the truncated ADH1 promoter using an immunoblot analysis was not applicable.

The MY1HS screens were successful in identifying several TGA2.2-interacting proteins (Table 6.1 and Table 6.1). In this regard, it might be significant to mention that two types of cDNA clones were expected to activate reporter gene expression: those that bind directly to the as-1 element and those that interact directly with TGA2.2 but do

not bind to the as-1 elements. In the MY1HS screens using the tobacco cDNA library,

two TGA transcription factor-related cDNAs, TGA2.1 and TGA10 were isolated (Table 6.2). The observation that a TGA10 cDNA clone was isolated in the MY1HS using a cDNA library prepared from tobacco leaves suggests that the TGA10 mRNA might be expressed in vegetative tissues. However, RT-PCR and protein immunoblot expression analysis demonstrated that TGA10 is a root specific TGA transcription factor

(Schiermeyer et al., 2003). Even though, it is necessary to conduct an independent expression analysis to confirm these results.

The MY1HS screens had identified other candidates that can interact with TGA2.2 without binding to the as-1 element. A screen with an Arabidopsis cDNA library yielded four putative TGA2.2-interacting candidates (Table 6.1). However, there were distinct and overlapping interaction patterns among the four putative candidates with the TGA transcription factors. For instance, the Y2HS experiments revealed that the isolated At4g00270 cDNA clone did not interacted with TGA2.2 prey protein when it was fused to the GAL4BD domain (data not shown). Among the isolated TGA2.2 interacting proteins, the focus was concentrated on the characterizing of two cDNA groups, groups 3 and 4, respectively (Table 6.1 and Table 6.1).

Group 3 cDNA inserts, encoding the At1g50570 and At5g55530 proteins, respectively, were selected for multiple reasons. First, both cDNA inserts encode for

two homologous proteins. Second, the presence of the conserved C2 domain, which is

involved in a variety of signal transduction pathways. Finally, the group interacts

exclusively with the class-II of TGA factors. The group 4 cDNA insert, encoding the At1g28480 protein, was selected because of its ability to interact with all identified TGA factors, sequence similarity to glutaredoxins and its potential ability to mediate changes of the redox state of TGA factors.