SGT1 positively regulates the process of plant cell death during both compatible and incompatible plant-pathogen interactions

SGT1 (suppressor of G2 allele of Skp1), an interactor of SCF (Skp1-Cullin-F-box) ubiquitin ligase complexes that mediate protein degradation, plays an important role at both G1-S and G2-M cell cycle transitions in yeast, and is highly conserved throughout eukaryotes. Plant SGT1 is required for both resistance (R) gene-mediated disease resistance and nonhost resistance to certain pathogens. Using virus-induced gene silencing (VIGS) in Nicotiana benthamiana, we demonstrate that SGT1 positively regulates the process of cell death during both host and nonhost interactions with various pathovars of Pseudomonas syringae. Silencing of NbSGT1 in N. benthamiana plants delays the induction of hypersensitive response (HR)-mediated cell death against nonhost pathogens and the development of disease-associated cell death caused by the host pathogen P. syringae pv. tabaci. Our results further demonstrate that NbSGT1 is required for Erwinia carotovora- and Sclerotinia sclerotiorum-induced disease-associated cell death. Overexpression of NbSGT1 in N. benthamiana accelerates the development of HR during R gene-mediated disease resistance and nonhost resistance. Our data also indicate that SGT1 is required for pathogen-induced cell death, but is not always necessary for the restriction of bacterial multiplication in planta. Therefore, we conclude that SGT1 is an essential component affecting the process of cell death during both compatible and incompatible plant-pathogen interactions. © 2010 The Authors

Jasmonate ZIM-Domain (JAZ) Protein Regulates Host and Nonhost Pathogen-Induced Cell Death in Tomato and <i>Nicotiana benthamiana</i>

<div><p>The nonhost-specific phytotoxin coronatine (COR) produced by several pathovars of <i>Pseudomonas syringae</i> functions as a jasmonic acid-isoleucine (JA-Ile) mimic and contributes to disease development by suppressing plant defense responses and inducing reactive oxygen species in chloroplast. It has been shown that the F-box protein CORONATINE INSENSITIVE 1 (COI1) is the receptor for COR and JA-Ile. JASMONATE ZIM DOMAIN (JAZ) proteins act as negative regulators for JA signaling in <i>Arabidopsis</i>. However, the physiological significance of JAZ proteins in <i>P. syringae</i> disease development and nonhost pathogen-induced hypersensitive response (HR) cell death is not completely understood. In this study, we identified <i>JAZ</i> genes from tomato, a host plant for <i>P. syringae</i> pv. <i>tomato</i> DC3000 (<i>Pst</i> DC3000), and examined their expression profiles in response to COR and pathogens. Most <i>JAZ</i> genes were induced by COR treatment or inoculation with COR-producing <i>Pst</i> DC3000, but not by the COR-defective mutant DB29. Tomato SlJAZ2, SlJAZ6 and SlJAZ7 interacted with SlCOI1 in a COR-dependent manner. Using virus-induced gene silencing (VIGS), we demonstrated that SlJAZ2, SlJAZ6 and SlJAZ7 have no effect on COR-induced chlorosis in tomato and <i>Nicotiana benthamiana</i>. However, <i>SlJAZ2</i>-, <i>SlJAZ6</i>- and <i>SlJAZ7</i>-silenced tomato plants showed enhanced disease-associated cell death to <i>Pst</i> DC3000. Furthermore, we found delayed HR cell death in response to the nonhost pathogen <i>Pst</i> T1 or a pathogen-associated molecular pattern (PAMP), INF1, in <i>SlJAZ2</i>- and <i>SlJAZ6</i>-silenced <i>N. benthamiana</i>. These results suggest that tomato JAZ proteins regulate the progression of cell death during host and nonhost interactions.</p></div

COR-dependent interaction between SlCOI1 and SlJAZs.

<p>(<b>A</b>) Interaction between SlCOI1 and SlJAZs. (<b>B</b>) Interaction between SlNINJA and SlJAZs. The MaV203 yeast strain carrying the construct for SlCOI1 (bait) and SlJAZs (prey) was grown on synthetic dropout (SD) glucose medium without Leu, Trp and His in the absence or presence of COR (20 µM). Photographs were taken at 5 days.</p

Involvement of SGT1 in COR-mediated signal transduction pathway leading to disease symptom development

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), that causes bacterial speck disease on tomato, produces a non-host-specific virulence effector, coronatine (COR). COR functions as a jasmonic acid (JA)-isoleucine mimic in planta and has multiple roles in the pathogenicity of Pst DC3000. One of the hallmarks of bacterial speck disease on tomato is the formation of necrotic lesions surrounded by chlorosis and COR is required for disease development. However, the molecular basis of COR-mediated disease symptom development including chlorosis and necrosis is still largely unknown. In our recent publication in New Phytologist, using virus-induced gene silencing (VIGS) based reverse genetics screen, we demonstrated that SGT1 (suppressor of G2 allele of skp1) is required for COR-induced chlorosis in Nicotiana benthamiana. SGT1-silenced tomato leaves showed a complete loss of COR-induced chlorosis and reduced disease symptom development after the inoculation with Pst DC3000. Furthermore, Arabidopsis sgt1b mutant was less sensitive to COR-induced root growth inhibition and showed delayed Pst DC3000 disease symptoms. In this addendum, we discuss the possible contribution of SGT1 to COR-mediated signal transduction pathway leading to disease symptom development during Pst DC3000 pathogenesis in tomato and Arabidopsis

Expression profiles of <i>JAZ</i> genes in tomato seedlings inoculated with <i>P. syringae</i> pv. <i>tomato</i> DC3000 (<i>Pst</i> DC3K) or the COR-defective mutant <i>Pst</i> DB29, or treated with COR.

<p>Tomato seedlings were treated with distilled water (mock control), COR (20 pmol) or inoculated with <i>Pst</i> DC3K or <i>Pst</i> DB29. The expression of genes encoding tomato JAZs was evaluated by real-time quantitative RT-PCR. Bars represent the means ± standard deviation (SD).</p

A Virus-Induced Gene Silencing Screen Identifies a Role for Thylakoid Formation1 in Pseudomonas syringae pv tomato Symptom Development in Tomato and Arabidopsis1[W][OA]

Pseudomonas syringae pv tomato DC3000 (Pst DC3000), which causes disease in tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana), produces coronatine (COR), a non-host-specific phytotoxin. COR, which functions as a jasmonate mimic, is required for full virulence of Pst DC3000 and for the induction of chlorosis in host plants. Previous genetic screens based on insensitivity to COR and/or methyl jasmonate identified several potential targets for COR and methyl jasmonate. In this study, we utilized Nicotiana benthamiana and virus-induced gene silencing to individually reduce the expression of over 4,000 genes. The silenced lines of N. benthamiana were then screened for altered responses to purified COR. Using this forward genetics approach, several genes were identified with altered responses to COR. These were designated as ALC (for altered COR response) genes. When silenced, one of the identified genes, ALC1, produced a hypersensitive/necrosis-like phenotype upon COR application in a Coronatine-Insensitive1 (COI1)-dependent manner. To understand the involvement of ALC1 during the Pst DC3000-host interaction, we used the nucleotide sequence of ALC1 and identified its ortholog in Arabidopsis (Thylakoid Formation1 [THF1]) and tomato (SlALC1). In pathogenicity assays performed on Arabidopsis thf1 mutant and SlALC1-silenced tomato plants, Pst DC3000 induced accelerated coalescing necrotic lesions. Furthermore, we showed that COR affects ALC1 localization in chloroplasts in a COI1-dependent manner. In conclusion, our results show that the virus-induced gene silencing-based forward genetic screen has the potential to identify new players in COR signaling and disease-associated necrotic cell death