An importin alpha homolog, MOS6, plays an important role in plant innate immunity

Plant disease resistance is the consequence of an innate defense mechanism mediated by Resistance (R) genes [1]. The conserved structure of one class of R protein is reminiscent of Toll-like receptors (TLRs) and Nucleotide binding oligomerization domain (NOD) proteins-immune-response perception modules in animal cells [2, 3, and 4]. The Arabidopsis snc1 (suppressor of npr1-1, constitutive, 1) mutant contains a mutation in a TIR-NBS-LRR-type of R gene that renders resistance responses constitutively active without interaction with pathogens [5]. Few components of the downstream signaling network activated by snc1 are known. To search for regulators of R-gene-mediated resistance, we screened for genetic suppressors of snc1. Three alleles of the mutant mos6 (modifier of snc1, 6) partially suppressed constitutive-resistance responses and immunity to virulent pathogens in snc1. Furthermore, the mos6-1 single mutant exhibited enhanced disease susceptibility to a virulent oomycete pathogen. MOS6, identified by positional cloning, encodes importin alpha3, one of eight alpha importins in Arabidopsis [6]. alpha importins mediate the import of specific proteins across the nuclear envelope. We previously reported that MOS3, a protein homologous to human nucleoporin 96, is required for constitutive resistance in snc1 [7]. Our data highlight an essential role for nucleo-cytoplasmic trafficking, especially protein import, in plant innate immunity.     

MOS2, a protein containing G-patch and KOW motifs, is essential for innate immunity in Arabidopsis thaliana

Innate immunity is critical for sensing and defending against microbial infections in multicellular organisms. In plants, disease resistance genes (R genes) play central roles in recognizing pathogens and initiating downstream defense cascades. Arabidopsis SNC1 encodes a TIR-NBS-LRR-type R protein with a similar structure to nucleotide binding oligomerization domain (Nod) proteins in animals. A point mutation in the region between the NBS and LRR of SNC1 results in constitutive activation of defense responses in the snc1 mutant. Here, we report the identification and characterization of mos2-1, a mutant suppressing the constitutive defense responses in snc1. Analysis of mos2 single mutants indicated that it is not only required for resistance specified by multiple R genes, but also for basal resistance. Map-based cloning of MOS2 revealed that it encodes a novel nuclear protein that contains one G-patch and two KOW domains and has homologs across the animal kingdom. The presence of both G-patch and KOW domains in the MOS2 protein suggests that it probably functions as an RNA binding protein critical for plant innate immunity. Our discovery on the biological functions of MOS2 will shed light on functions of the MOS2 homologs in animals, where they may also play important roles in innate immunity.     

A putative nucleoporin 96 Is required for both basal defense and constitutive resistance responses mediated by suppressor of npr1-1,constitutive 1

The Arabidopsis thaliana suppressor of npr1-1, constitutive 1 (snc1) mutant contains a gain-of-function mutation in a Toll Interleukin1 receptor-nucleotide binding-Leu-rich repeat-type resistance gene (R-gene), which leads to constitutive activation of disease resistance response against pathogens. In a screen for suppressors of snc1, a recessive mutation, designated mos3 (for modifier of snc1,3), was found to suppress the constitutive pathogenesis-related gene expression and resistance to virulent Pseudomonas syringae maculicola ES4326 and Peronospora parasitica Noco2 in snc1. In addition, mos3 is also compromised in resistance mediated by Resistance to Peronospora parasitica 4 (RPP4), Resistance to Pseudomonas syringae pv maculicola (RPM1), and Resistance to Pseudomonas syringae 4 (RPS4). Single mutant mos3 plants exhibited enhanced disease susceptibility to P. s. pv maculicola ES4326, suggesting that MOS3 is required for basal resistance to pathogens as well. mos3-1 was identified by map-based cloning, and it encodes a protein with high sequence similarity to human nucleoporin 96. Localization of the MOS3-green fluorescent protein fusion to the nuclear envelope further indicates that MOS3 may encode a nucleoporin, suggesting that nuclear and cytoplasmic trafficking plays an important role in both R-gene-mediated and basal disease resistance.     

Activation of an EDS1-mediated R-gene pathway in the snc1 mutant leads to constitutive, NPR1-independent pathogen resistance

The Arabidopsis NPR1 protein is an essential regulatory component of systemic acquired resistance (SAR). Mutations in the NPR1 gene completely block the induction of SAR by signals such as salicylic acid (SA). An Arabidopsis mutant, snc1 (suppressor of npr1-1, constitutive 1), was isolated in a screen for suppressors of npr1-1. In the npr1-1 background, the snc1 mutation resulted in constitutive resistance to Pseudomonas syringae maculicola ES4326 and Peronospora parasitica Noco2. High levels of SA were detected in the mutant and shown to be required for manifestation of the snc1 phenotype. The snc1 mutation was mapped to the RPP5 resistance (R) gene cluster and the eds1 mutation that blocks RPP5-mediated resistance suppressed snc1. These data suggest that a RPP5-related resistance pathway is activated constitutively in snc1. This pathway does not employ NPR1 but requires the signal molecule SA and the function of EDS1. Moreover, in snc1, constitutive resistance is conferred in the absence of cell death, which is often associated with R-gene mediated resistance.     

A novel role for protein farnesylation in plant innate immunity

Plants utilize tightly regulated mechanisms to defend themselves against pathogens. Initial recognition results in activation of specific Resistance (R) proteins that trigger downstream immune responses, in which the signaling networks remain largely unknown. A point mutation in SUPPRESSOR OF NPR1 CONSTITUTIVE1 (SNC1), a RESISTANCE TO PERONOSPORA PARASITICA4 R gene homolog, renders plants constitutively resistant to virulent pathogens. Genetic suppressors of snc1 may carry mutations in genes encoding novel signaling components downstream of activated R proteins. One such suppressor was identified as a novel loss-of-function allele of ENHANCED RESPONSE TO ABSCISIC ACID1 (ERA1), which encodes the beta-subunit of protein farnesyltransferase. Protein farnesylation involves attachment of C15-prenyl residues to the carboxyl termini of specific target proteins. Mutant era1 plants display enhanced susceptibility to virulent bacterial and oomycete pathogens, implying a role for farnesylation in basal defense. In addition to its role in snc1-mediated resistance, era1 affects several other R-protein-mediated resistance responses against bacteria and oomycetes. ERA1 acts partly independent of abscisic acid and additively with the resistance regulator NON-EXPRESSOR OF PR GENES1 in the signaling network. Defects in geranylgeranyl transferase I, a protein modification similar to farnesylation, do not affect resistance responses, indicating that farnesylation is most likely specifically required in plant defense signaling. Taken together, we present a novel role for farnesyltransferase in plant-pathogen interactions, suggesting the importance of protein farnesylation, which contributes to the specificity and efficacy of signal transduction events.     

The Ankyrin-Repeat Transmembrane Protein BDA1 Functions Downstream of the Receptor-Like Protein SNC2 to Regulate Plant Immunity

Plants utilize a large number of immune receptors to recognize pathogens and activate defense responses. A small number of these receptors belong to the receptor-like protein family. Previously, we showed that a gain-of-function mutation in the receptor-like protein SNC2 (for Suppressor of NPR1, Constitutive2) leads to constitutive activation of defense responses in snc2-1D mutant plants. To identify defense signaling components downstream of SNC2, we carried out a suppressor screen in the snc2-1D mutant background of Arabidopsis (Arabidopsis thaliana). Map-based cloning of one of the suppressor genes, BDA1 (for bian da; "becoming big" in Chinese), showed that it encodes a protein with amino-terminal ankyrin repeats and carboxyl-terminal transmembrane domains. Loss-of-function mutations in BDA1 suppress the dwarf morphology and constitutive defense responses in snc2-1D npr1-1 (for nonexpressor of pathogenesis-related genes1,1) and also result in enhanced susceptibility to bacterial pathogens. In contrast, a gain-of-function allele of bda1 isolated from a separate genetic screen to search for mutants with enhanced pathogen resistance was found to constitutively activate cell death and defense responses. These data suggest that BDA1 is a critical signaling component that functions downstream of SNC2 to regulate plant immunity.     

Arabidopsis snc2-1D Activates Receptor-Like Protein-Mediated Immunity Transduced through WRKY70

Plant immune receptors belonging to the receptor-like protein (RLP) family contain extracellular leucine-rich repeats (LRRs) and a short cytoplasmic tail linked by a single transmembrane motif. Here, we report the identification of snc2-1D (for suppressor of npr1-1, constitutive 2), a semidominant Arabidopsis thaliana mutant with constitutively activated defense responses. Map-based cloning of snc2-1D showed that it encodes an RLP. The point mutation in snc2-1D leads to substitution of the second Gly for Arg in the conserved GXXXG motif of the transmembrane helix, suggesting that this residue is important for negative regulation of the protein. Epistasis analysis revealed that the snc2-1D mutant phenotype is not affected by mutations in genes known to be required for the nucleotide binding (NB)-LRR Resistance (R) protein signaling. A suppressor screen of snc2-1D was performed, and map-based cloning of one suppressor revealed that mutations in WRKY70 suppress the constitutive defense responses in snc2-1D, suggesting that WRKY70 functions downstream of snc2-1D. The identification of snc2-1D provides us with a unique system for genetic analysis of resistance pathways downstream of RLPs, which may be distinct from those downstream of NB-LRR type R proteins.     

The ubiquitin pathway is required for innate immunity in Arabidopsis

Plant defences require a multitude of tightly regulated resistance responses. In Arabidopsis, the unique gain-of-function mutant suppressor of npr1-1 constitutive 1 ( snc1 ) carries a point mutation in a Resistance ( R )-gene, resulting in constitutive activation of defence responses without interaction with pathogens. This has allowed us to identify various downstream signalling components essential in multiple defence pathways. One mutant that suppresses snc1 -mediated constitutive resistance is modifier of snc1 5 ( mos5 ), which carries a 15-bp deletion in UBA1 , one of two ubiquitin-activating enzyme genes in Arabidopsis. A mutation in UBA2 does not suppress snc1 , suggesting that these two genes are not equally required in Arabidopsis disease resistance. On the other hand, a mos5 uba2 double mutant is lethal, implying partial redundancy of the two homologues. Apart from affecting snc1 -mediated resistance, mos5 also exhibits enhanced disease susceptibility to a virulent pathogen and is impaired in response to infection with avirulent bacteria carrying the protease elicitor AvrRpt2. The mos5 mutation in the C-terminus of UBA1 might affect binding affinity of the downstream ubiquitin-conjugating enzymes, thus perturbing ubiquitination of target proteins. Furthermore, SGT1b and RAR1, which are necessary for resistance conferred by the SNC1 -related R -genes RPP4 and RPP5 , are dispensable in snc1 -mediated resistance. Our data reveal the definite requirement for the ubiquitination pathway in the activation and downstream signalling of several R-proteins.     

Transportin-SR is required for proper splicing of resistance genes and plant immunity

Transportin-SR (TRN-SR) is a member of the importin-β super-family that functions as the nuclear import receptor for serine-arginine rich (SR) proteins, which play diverse roles in RNA metabolism. Here we report the identification and cloning of mos14 (modifier of snc1-1, 14), a mutation that suppresses the immune responses conditioned by the auto-activated Resistance (R) protein snc1 (suppressor of npr1-1, constitutive 1). MOS14 encodes a nuclear protein with high similarity to previously characterized TRN-SR proteins in animals. Yeast two-hybrid assays showed that MOS14 interacts with AtRAN1 via its N-terminus and SR proteins via its C-terminus. In mos14-1, localization of several SR proteins to the nucleus was impaired, confirming that MOS14 functions as a TRN-SR. The mos14-1 mutation results in altered splicing patterns of SNC1 and another R gene RPS4 and compromised resistance mediated by snc1 and RPS4, suggesting that nuclear import of SR proteins by MOS14 is required for proper splicing of these two R genes and is important for their functions in plant immunity.     

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.     

Evolution of a complex disease resistance gene cluster in diploid Phaseolus and tetraploid Glycine

We used a comparative genomics approach to investigate the evolution of a complex nucleotide-binding (NB)-leucine-rich repeat (LRR) gene cluster found in soybean (Glycine max) and common bean (Phaseolus vulgaris) that is associated with several disease resistance (R) genes of known function, including Rpg1b (for Resistance to Pseudomonas glycinea1b), an R gene effective against specific races of bacterial blight. Analysis of domains revealed that the amino-terminal coiled-coil (CC) domain, central nucleotide-binding domain (NB-ARC [for APAF1, Resistance genes, and CED4]), and carboxyl-terminal LRR domain have undergone distinct evolutionary paths. Sequence exchanges within the NB-ARC domain were rare. In contrast, interparalogue exchanges involving the CC and LRR domains were common, consistent with both of these regions coevolving with pathogens. Residues under positive selection were overrepresented within the predicted solvent-exposed face of the LRR domain, although several also were detected within the CC and NB-ARC domains. Superimposition of these latter residues onto predicted tertiary structures revealed that the majority are located on the surface, suggestive of a role in interactions with other domains or proteins. Following polyploidy in the Glycine lineage, NB-LRR genes have been preferentially lost from one of the duplicated chromosomes (homeologues found in soybean), and there has been partitioning of NB-LRR clades between the two homeologues. The single orthologous region in common bean contains approximately the same number of paralogues as found in the two soybean homeologues combined. We conclude that while polyploidization in Glycine has not driven a stable increase in family size for NB-LRR genes, it has generated two recombinationally isolated clusters, one of which appears to be in the process of decay.     

Constitutive disease resistance requires EDS1 in the Arabidopsis mutants cpr1 and cpr6 and is partially EDS1-dependent in cpr5

The systemic acquired resistance (SAR) response in Arabidopsis is characterized by the accumulation of salicylic acid (SA), expression of the pathogenesis-related (PR) genes, and enhanced resistance to virulent bacterial and oomycete pathogens. The cpr (constitutive expressor of PR genes) mutants express all three SAR phenotypes. In addition, cpr5 and cpr6 induce expression of PDF1.2, a defense-related gene associated with activation of the jasmonate/ethylene-mediated resistance pathways. cpr5 also forms spontaneous lesions. In contrast, the eds1 (enhanced disease susceptibility) mutation abolishes race-specific resistance conferred by a major subclass of resistance (R) gene products in response to avirulent pathogens. eds1 plants also exhibit increased susceptibility to virulent pathogens. Epistasis experiments were designed to explore the relationship between the cpr- and EDS1-mediated resistance pathways. We found that a null eds1 mutation suppresses the disease resistance phenotypes of both cpr1 and cpr6. In contrast, eds1 only partially suppresses resistance in cpr5, leading us to conclude that cpr5 expresses both EDS1-dependent and EDS1-independent components of plant disease resistance. Although eds1 does not prevent lesion formation on cpr5 leaves, it alters their appearance and reduces their spread. This phenotypic difference is associated with increased pathogen colonization of cpr5 eds1 plants compared to cpr5. The data allow us to place EDS1 as a necessary downstream component of cpr1- and cpr6-mediated responses, but suggest a more complex relationship between EDS1 and cpr5 in plant defense.     

EDS1 in tomato is required for resistance mediated by TIR-class R genes and the receptor-like R gene Ve

In tobacco and other Solanaceae species, the tobacco N gene confers resistance to tobacco mosaic virus (TMV), and leads to induction of standard defense and resistance responses. Here, we report the use of N-transgenic tomato to identify a fast-neutron mutant, sun1-1 (suppressor of N), that is defective in N-mediated resistance. Induction of salicylic acid (SA) and expression of pathogenesis-related (PR) genes, each signatures of systemic acquired resistance, are both dramatically suppressed in sun1-1 plants after TMV treatment compared to wild-type plants. Application of exogenous SA restores PR gene expression, indicating that SUN1 acts upstream of SA. Upon challenge with additional pathogens, we found that the sun1-1 mutation impairs resistance mediated by certain resistance (R) genes, (Bs4, I, and Ve), but not others (Mi-1). In addition, sun1-1 plants exhibit enhanced susceptibility to TMV, as well as to virulent pathogens. sun1-1 has been identified as an EDS1 homolog present on chromosome 6 of tomato. The discovery of enhanced susceptibility in the sun1-1 (Le_eds1-1) mutant plant, which contrasts to reports in Nicotiana benthamiana using virus-induced gene silencing, provides evidence that the intersection of R gene-mediated pathways with general resistance pathways is conserved in a Solanaceous species. In tomato, EDS1 is important for mediating resistance to a broad range of pathogens (viral, bacterial, and fungal pathogens), yet shows specificity in the class of R genes that it affects (TIR-NBS-LRR as opposed to CC-NBS-LRR). In addition, a requirement for EDS1 for Ve-mediated resistance in tomato exposes that the receptor-like R gene class may also require EDS1.