A single amino acid insertion in the WRKY domain of the Arabidopsis TIR-NBS-LRR-WRKY-type disease resistance protein SLH1 (sensitive to low humidity 1) causes activation of defense responses and hypersensitive cell death.

In this study we characterized the sensitive to low humidity 1 (slh1) mutant of Arabidopsis ecotype No-0 which exhibits normal growth on agar plate medium but which on transfer to soil shows growth arrest and development of necrotic lesions. cDNA microarray hybridization and RNA gel blot analysis revealed that genes associated with activation of disease resistance were upregulated in the slh1 mutants in response to conditions of low humidity. Furthermore, the slh1 mutants accumulate callose, autofluorescent compounds and salicylic acid (SA). We demonstrate that SA is required for the slh1 phenotype but not PAD4 or NPR1. SLH1 was isolated by map-based cloning and it encodes a resistance (R)-like protein consisting of a domain with Toll and interleukin-1 receptor homology (TIR), a nucleotide-binding domain (NB), leucine-rich repeats (LRR) and a carboxy-terminal WRKY domain. SLH1 is identical to the R gene RRS1-R of the Arabidopsis ecotype Nd-1, a gene which confers resistance to the bacterial pathogen Ralstonia solanacearum GMI1000 and also functions as an R gene to this pathogen in No-0. We identified a 3 bp insertion mutation in slh1 that results in the addition of a single amino acid in the WRKY domain; thereby impairing its DNA-binding activity. Our data suggest that SLH1 disease resistance signaling may be negatively regulated by its WRKY domain in the R protein and that the constitutive defense activation conferred by the slh1 mutation is inhibited by conditions of high humidity.

Noutoshi Y ，Ito T ，Seki M ，Nakashita H ，Yoshida S ，Marco Y ，Shirasu K ，Shinozaki K ... -  《PLANT JOURNAL》

High humidity suppresses ssi4-mediated cell death and disease resistance upstream of MAP kinase activation, H2O2 production and defense gene expression.

The Arabidopsis ssi4 mutant, which exhibits spontaneous lesion formation, constitutive expression of pathogenesis-related (PR) genes and enhanced resistance to virulent bacterial and oomycete pathogens, contains a gain-of-function mutation in a TIR-NBS-LRR type R gene. Epistatic analyses revealed that both PR gene expression and disease resistance are activated via a salicylic acid (SA)- and EDS1-dependent, but NPR1- and NDR1-independent signaling pathway. In this study, we demonstrate that in moderate relative humidity (RH; 60%), the ssi4 mutant accumulates H(2)O(2) and SA prior to lesion formation and displays constitutive activation of the MAP kinases AtMPK6 and AtMPK3. It also constitutively expresses a variety of defense-associated genes, including those encoding the WRKY transcription factors AtWRKY29 and AtWRKY6, the MAP kinases AtMPK6 and AtMPK3, the powdery mildew R proteins RPW8.1 and RPW8.2, EDS1 and PR proteins. All of these ssi4-induced responses, as well as the chlorotic, stunted morphology and enhanced disease resistance phenotype, are suppressed by high RH (95%) growth conditions. Thus, a humidity sensitive factor (HSF) appears to function at an early point in the ssi4 signaling pathway. All ssi4 phenotypes, except for MAP kinase activation, also were suppressed by the eds1-1 mutation. Thus, ssi4-induced MAP kinase activation occurs downstream of the HSF but either upstream of EDS1 or on a separate branch of the ssi4 signaling pathway. SA is a critical signaling component in ssi4-mediated defense responses. However, exogenously supplied SA failed to restore lesion formation in high RH-grown ssi4 plants, although it induced defense gene expression. Thus, additional signals also are involved.

Zhou F ，Menke FL ，Yoshioka K ，Moder W ，Shirano Y ，Klessig DF ... -  《PLANT JOURNAL》

Signaling requirements and role of salicylic acid in HRT- and rrt-mediated resistance to turnip crinkle virus in Arabidopsis.

Inoculation of turnip crinkle virus (TCV) on the resistant Arabidopsis ecotype Di-17 elicits a hypersensitive response (HR), which is accompanied by increased expression of pathogenesis-related (PR) genes. Previous genetic analyses revealed that the HR to TCV is conferred by HRT, which encodes a coiled-coil (CC), nucleotide-binding site (NBS) and leucine-rich repeat (LRR) class resistance (R) protein. In contrast to the HR, resistance to TCV requires both HRT and a recessive allele at a second locus designated rrt. Here, we demonstrate that unlike most CC-NBS-LRR R genes, HRT/rrt-mediated resistance is dependent on EDS1 and independent of NDR1. Resistance is also independent of RAR1 and SGT1. HRT/rrt-mediated resistance is compromised in plants with reduced salicylic acid (SA) content as a consequence of mutations eds5, pad4, or sid2. By contrast, HR is not affected by mutations in eds1, eds5, pad4, sid2, ndr1, rar1, or sgt1b. Resistance to TCV is restored in both SA-deficient Di-17 plants expressing the nahG transgene and mutants containing the eds1, eds5, or sid2 mutations by exogenous application of SA or the SA analog benzo(1,2,3)thiadiazole-7-carbothioic acid (BTH). In contrast, SA/BTH treatment failed to enhance resistance in HRT pad4, Col-0, or hrt homozygous progeny of a cross between Di-17 and Col-0. Thus, HRT and PAD4 are required for SA-induced resistance. Exogenously supplied SA or high endogenous levels of SA, due to the ssi2 mutation, overcame the suppressive effects of RRT and enhanced resistance to TCV, provided the HRT allele was present. High levels of SA upregulate HRT expression via a PAD4-dependent pathway. As Col-0 transgenic lines expressing high levels of HRT were resistant to TCV, but lines expressing moderate to low levels of HRT were not, we conclude that SA enhances resistance in the RRT background by upregulating HRT expression. These data suggest that the HRT-TCV interaction is unable to generate sufficient amounts of SA required for a stable resistance phenotype, and the presence of rrt possibly corrects this deficiency.

Chandra-Shekara AC ，Navarre D ，Kachroo A ，Kang HG ，Klessig D ，Kachroo P ... -  《PLANT JOURNAL》

Two TIR:NB:LRR genes are required to specify resistance to Peronospora parasitica isolate Cala2 in Arabidopsis.

Resistance responses that plants deploy in defence against pathogens are often triggered following a recognition event mediated by resistance (R) genes. The encoded R proteins usually contain a nucleotide-binding site (NB) and a leucine-rich repeat (LRR) domain. They are further classified into those that contain an N-terminal coiled coil (CC) motif or a Toll interleukin receptor (TIR) domain. Such R genes, when transferred into a susceptible plant of the same or closely related species, usually impart full resistance capability. We have used map-based cloning and mutation analysis to study the recognition of Peronospora parasitica (RPP)2 (At) locus in Arabidopsis accession Columbia (Col-0), which is a determinant of specific recognition of P. parasitica (At) isolate Cala2. Genetic mapping located RPP2 to a 200-kb interval on chromosome 4, which contained four adjacent TIR:NB:LRR genes. Mutational analysis revealed three classes of genes involved in specifying resistance to Cala2. One class, which resulted in pleiotropic effects on resistance to other P. parasitica (At) isolates, was unlinked to the RPP2 locus; this class included AtSGT1b. The other two classes were mapped within the interval and were specific to Cala2 resistance. Representatives of each of these classes were sequenced, and mutations were found in one or the other of two (RPP2A and RPP2B) of the four TIR:NB:LRR genes. RPP2A and RPP2B complemented their specific mutations, but failed to impart resistance when present alone, and it is concluded that both genes are essential determinants for isolate-specific recognition of Cala2. RPP2A has an unusual structure with a short LRR domain at the C-terminus, preceded by two potential but incomplete TIR:NB domains. In addition, the RPP2A LRR domain lacks conserved motifs found in all but three other TIR:NB:LRR class proteins. In contrast, RPP2B has a complete TIR:NB:LRR structure. It is concluded that RPP2A and RPP2B cooperate to specify Cala2 resistance by providing recognition or signalling functions lacked by either partner protein.

Sinapidou E ，Williams K ，Nott L ，Bahkt S ，Tör M ，Crute I ，Bittner-Eddy P ，Beynon J ... -  《PLANT JOURNAL》

The atypical resistance gene, RPW8, recruits components of basal defence for powdery mildew resistance in Arabidopsis.

Genetic studies have identified a number of components of signal transduction pathways leading to plant disease resistance and the accompanying hypersensitive response (HR) following detection of pathogens by plant resistance (R) genes. In Arabidopsis, the majority of R proteins so far characterized belong to a plant superfamily that have a central nucleotide-binding site and C-terminal leucine-rich-repeats (NB-LRRs). Another much less prevalent class comprises RPW8.1 and RPW8.2, two related proteins that possess a putative N-terminal transmembrane domain and a coiled-coil motif, and confer broad-spectrum resistance to powdery mildew. Here we investigated whether RPW8.1 and RPW8.2 engage known pathway(s) for defence signalling. We show that RPW8.1 and RPW8.2 recruit, in addition to salicylic acid and EDS1, the other NB-LRR gene-signalling components PAD4, EDS5, NPR1 and SGT1b for activation of powdery mildew resistance and HR. In contrast, NDR1, RAR1 and PBS3 that are required for function of certain NB-LRR R genes, and COI1 and EIN2 that operate, respectively, in the jasmonic acid and ethylene signalling pathways, do not contribute to RPW8.1 and RPW8.2-mediated resistance. We further demonstrate that EDR1, a gene encoding a conserved MAPKK kinase, exerts negative regulation on HR cell death and powdery mildew resistance by limiting the transcriptional amplification of RPW8.1 and RPW8.2. Our results suggest that RPW8.1 and RPW8.2 stimulate a conserved basal defence pathway that is negatively regulated by EDR1.

Xiao S ，Calis O ，Patrick E ，Zhang G ，Charoenwattana P ，Muskett P ，Parker JE ，Turner JG ... -  《PLANT JOURNAL》