Strong Suppression of Systemic Acquired Resistance in Arabidopsis by NRR is Dependent on its Ability to Interact with NPR1and its Putative Repression Domain

Mawsheng Chern 0 Patrick E. Canlas 0 Pamela C. Ronald 0 0 Department of Plant Pathology, University of California , Davis, CA 95616, USA Systemic Acquired Resistance (SAR) in plants confers lasting broad-spectrum resistance to pathogens and requires the phytohormone salicylic acid (SA). Arabidopsis NPR1/NIM1 is a key regulator of the SAR response. Studies attempting to reveal the function of NPR1 and how it mediates SA signaling have led to isolation of two classes of proteins that interact with NPR1: the first class includes rice NRR, Arabidopsis NIMIN1, NIMIN2, and NIMIN3, and tobacco NIMIN2-like proteins; the second class belongs to TGA transcription factors. We have previously shown that overexpression of NRR in rice suppresses both basal and Xa21-mediated resistance. In order to test whether NRR affects SA-induced, NPR1-mediated SAR, we have transformed Arabidopsis with the rice NRR gene and tested its effects on the defense response. Expression of NRR in Arabidopsis results in suppression of PR gene induction by SAR inducer and resistance to pathogens. These phenotypes are even more severe than those of the npr1-1 mutant. The ability of NRR to suppress PR gene induction and disease resistance is correlated with its ability to bind to NPR1 because two point mutations in NRR, which reduce NPR1 binding, fail to suppress NPR1. In contrast, wild-type and a mutant NRR, which still binds to NPR1 strongly, retain the ability to suppress the SAR response. Replacing the C-terminal 79 amino acids of NRR with the VP16 activation domain turns the fusion protein into a transcriptional co-activator. These results indicate that NRR binds to NPR1 in vivo in a protein complex to inhibit transcriptional activation of PR genes and that NRR contains a transcription repression domain for active repression. - INTRODUCTION Systemic acquired resistance (SAR) is an induced defense response in plants; it induces expression of pathogenesis-related (PR) genes (Ryals et al., 1996) and confers lasting broadspectrum resistance to viral, bacterial, and fungal pathogens. In dicots, such as Arabidopsis and tobacco, the phytohormone salicylic acid (SA) and the synthetic chemicals 2,6-dichloroisonicotinic acid (INA) and benzothiadiazole (BTH) are potent inducers of SAR (Friedrich et al., 1996). The NPR1 (for nonexpresser of PR genes 1; also known as NIM1 and SAI1) gene is identified as a key regulator of the SA-mediated SAR pathway in Arabidopsis (Cao et al., 1994; Delaney et al., 1995; Glazebrook et al., 1996; Shah et al., 1997). NPR1 expression levels become elevated upon induction by SA, INA, BTH, or pathogen infection (Cao et al., 1997; Ryals et al., 1997). Arabidopsis npr1/nim1 mutants are impaired in their ability to induce PR gene expression and mount a SAR response, even after treatment with SA or INA. Intensive investigations have shed some light on how NPR1 mediates SAR. NPR1 contains a bipartite nuclear localization sequence and two potential proteinprotein interaction domains: an ankyrin repeat domain and a BTB/POZ domain (Cao et al., 1997; Ryals et al., 1997). NPR1 functions as a transcriptional co-activator in a TGA2NPR1 complex after SA treatment in an in-vivo transient cell assay; this function requires the BTB/POZ domain and the oxidation of NPR1 Cys-521 and Cys-529 (Rochon et al., 2006). Nuclear localization of NPR1 protein is essential for its function (Kinkema et al., 2000). Without induction, NPR1 protein forms an oligomer 1 To whom correspondence should be addressed. E-mail pcronald@ucdavis. edu, fax +1 530 752 5674. The Author 2008. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: 10.1093/mp/ssn017, Advance Access publication 22 April 2008 and is excluded from the nucleus. Redox changes mediate SAR induction, causing monomeric NPR1 to emerge and accumulate in the nucleus and activate PR gene expression (Mou et al. 2003). In search of proteins that mediate NPR1 function, several groups have identified TGA family members of basic-region leucine zipper (bZIP) transcription factors, both from Arabidopsis (Zhang et al., 1999; Despres et al., 2000; Zhou et al., 2000) and from rice (Chern et al., 2001), as NPR1 interacting proteins. The ankyrin repeats of NPR1 are necessary and sufficient for the interaction with TGA proteins but the interaction can be abolished by npr1-1 and npr1-2 mutants (Zhang et al., 1999). The interaction between NPR1 and TGA proteins facilitates in-vitro binding of the TGA proteins (Despres et al., 2000) and recruits them in vivo (Johnson et al., 2003) to the SAresponsive promoters. In-vivo interaction between NPR1 and a GAL4:TGA2 fusion (GAL4 DNA-binding domain fused to TGA2) protein leads to SA-mediated gene activation in Arabidopsis (Fan and Dong, 2002), supporting the notion that NPR1 binds in vivo to TGA2, which mediates transcriptional activation of downstream genes. The Arabidopsis triple knockout mutant tga2tga5tga6 blocks induction of PR gene expression and pathogen resistance (Zhang et al., 2003), further supporting the hypothesis that TGA proteins mediate NPR1 function. TGA2, TGA5, and TGA6 function redundantly as negative regulators of PR genes before induction (Zhang et al., 2003; Rochon et al., 2006). It is thought that, after induction, TGA proteins serve to anchor NPR1 to PR gene promoters to activate the genes. In Arabidopsis, another group of NIM1/NPR1 interacting proteins were identified and named NIMIN1, NIMIN2, and NIMIN3. These three Arabidopsis proteins share very limited sequence similarity but may be structurally related (Weigel et al., 2001). In tobacco, three NIMIN2-like (NIMIN2a, 2b, and 2c) were identified as NPR1 interactors (Zwicker et al., 2007). Weigel et al. (2005) further showed that overexpression of NIMIN1 in Arabidopsis led to abolishment of the SAR response after SA treatment and that knockout and RNAsilencing of NIMIN1 resulted in enhanced PR-1 gene expression after SA treatment, but no clear effects on disease resistance. In tobacco, Zwicker et al. (2007) showed that constitutive expression of NtNIMIN2a led to delayed PR-1 induction and suppression of NIMIN2 transcripts enhanced the accumulation of PR-1 protein. In Arabidopsis, overexpression of NPR1 leads to enhanced disease resistance to both bacterial and oomycete pathogens (Cao et al., 1998; Friedrich et al., 2001). In rice, overexpression of Arabidopsis NPR1 (Chern et al., 2001) or the rice homologue NH1 (Chern et al., 2005b) results in enhanced resistance to the pathogen Xanthomonas oryzae pv. oryzae (Xoo), strongly suggesting the presence of a related defense pathway in rice. We have previously reported the isolation and characterization of a novel rice gene NRR (for Negative Regulator of disease Resistance) (Chern et al., 2005a). Overexpression of NRR in rice leads to super-susceptibility to Xoo, impairing both basal and Xa21-mediated resistance. NRR int (...truncated)