Arabidopsis CAD1 negatively controls plant immunity mediated by both salicylic acid-dependent and -independent signaling pathways

Abstract

The Arabidopsis cad1 (constitutively activated cell death 1) mutant shows a phenotype of hypersensitive response (HR)-like cell death and increased levels of endogenous salicylic acid (SA), indicating that the CAD1 protein negatively controls the SA-mediated pathway in plant immunity. To clarify the relationship between the CAD1 protein and SA-mediated defense pathways, a molecular genetic analysis was carried out using mutants related to SA biosynthesis. A genetic cross between the cad1 mutant and the pad4 and sid2 mutants only partially suppressed the cad1 phenotypes. These double mutants still showed acquired resistance to Pst DC3000 infection similar to the cad1 single mutant. These results indicate that the CAD1 negatively controls not only the SA-dependent defense pathway but also the SA-independent pathway. To clarify the CAD1-mediated SA-independent pathway, a revertant was isolated from an M2 population of the sid2 cad1 mutant. The revertant can1t mutant did not show cell death phenotype and the plant and leaf size recovered to those of the wild-type. The can1t mutant also showed pathogen susceptibility comparable to wild type, indicating that the can1t mutant looses the pathogen-resistance that the cad1 mutant acquires. There results suggested that CAN1 is a positive regulator of the novel plant immunity under the control of CAD1.

Introduction

Plants have evolved various defense mechanisms to resist infection by pathogens. The host plant initiates multiple signal transduction pathways that activate various plant defenses and thereby limits pathogen growth. In many cases, the resistance is associated with increased expression of defense genes, including the pathogenesis-related (PR) genes and accumulation of salicylic acid (SA) in the infected leaf. SA has emerged as a key-signaling component that activates both hypersensitive response (HR) and PR gene expression [1], [2], [3]. When SA accumulation is suppressed in Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) by expression of the nahG transgene, which encodes the SA-degrading enzyme SA hydroxylase, susceptibility to both compatible and incompatible pathogens is enhanced and PR genes expression is suppressed [4], [5]. Several genes being important for SA accumulation in response to pathogen attack and for its transduction have been found. Positive regulators of SA production during infection by some pathogens include PAD4 (PHYTOALEXIN DEFICIENT 4), EDS1 (ENHANCED DISEASE SUSCEPTIBILITY 1), SAG101 (SNESCENCE-ASSOCIATED GENE 101) [6], [7], [8], [9], [10], and NDR1 (NON-RACE-SPECIFIC DISEASE RESISTANCE 1), a possible membrane protein [11], [12]. Furthermore, SID2 (SALICYLIC ACID INDUCTION DEFICIENT 2) is required for SA biosynthesis [13]. SID2/ICS1 encodes a pathogen-induced isochorismate synthase, and SA production is drastically reduced in sid2 mutants, indicating that the majority of SA in Arabidopsis is produced from isochorismate rather than from the alternative phenylalanine pathway [13], [14], [15], [16]. Thus, the sid2 mutant exhibits enhanced susceptibility to pathogen infection and impaired PR genes expression.

Lesion mimic mutants that result in constitutive misregulation of cell death are one of the tools with which to unravel the complex program cell death pathway, and a few such mutants have been identified, including acd (accelerated cell death), lsd (lesion simulating disease), cpr (constitutive expression of PR), ssi (suppressor of salicylic acid and insensitivity of npr1) and siz (SAP and Miz). Many lesion mimic mutants exhibit a state of increased disease resistance known as systemic acquired resistance (SAR) with constitutive activation of PR genes expression and the SA-signaling pathways [17], [18]. In mutants such as acd5, acd11, lsd6, lsd7, cpr22, ssi1 and siz1, spontaneous cell death and expression of PR1 gene is suppressed in the presence of the nahG transgene, supporting a crucial role for SA in lesion formation and expression of PR genes [17], [18]. These studies have demonstrated that these lesion mimic mutants show disruption of a gene that seems to be a negative regulator of the programmed cell death pathway in plant immunity. Despite the fact that powerful mutant screening in Arabidopsis has made significant contributions to identifying components of defense activation mechanisms, the overall view of these pathways remains unclear.

To clarify the processes involved in plant immunity, we have isolated and characterized a single recessive Arabidopsis mutant, cad1 (constitutively activated cell death 1), which has a phenotype that mimics the HR [19]. This mutant shows spontaneously activated expression of PR genes, leading to a 32-fold increase in SA. Inoculation of cad1 mutant plants with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) shows that the cad1 mutation results in a restriction of bacterial growth. Cloning of CAD1 reveals that this gene encodes a protein containing a domain with significant homology to the MACPF (membrane attack complex and perforin) domain of complement components and perforin proteins that are involved in innate immunity in animals. Furthermore, 35SnahG cad1 double mutant partially suppressed the cad1 phenotype. Thus, the CAD1 protein appears negatively to control the SA-mediated pathway of cell death in plant immunity [19], [20].

In this study, we carried out a molecular genetic analysis of SA biosynthesis-related and cad1 mutants. The results indicated that CAD1 negatively controls plant immunity mediated by distinct signaling pathways in both a salicylic acid-dependent and -independent manner.