Characterization of eds1, a mutation in Arabidopsis suppressing resistance to Peronospora parasitica specified by several different RPP genes.

The interaction between Arabidopsis and the biotrophic oomycete Peronospora parasitica (downy mildew) provides an attractive model pathosystem to identify molecular components of the host that are required for genotype-specific recognition of the parasite. These components are the so-called RPP genes (for resistance to P. parasitica). Mutational analysis of the ecotype Wassilewskija (Ws-0) revealed an RPP-nonspecific locus called EDS1 (for enhanced disease susceptibility) that is required for the function of RPP genes on chromosomes 3 (RPP1/RPP14 and RPP10) and 4 (RPP12). Genetic analyses demonstrated that the eds1 mutation is recessive and is not a defective allele of any known RPP gene, mapping to the bottom arm of chromosome 3 (approximately 13 centimorgans below RPP1/RPP14). Phenotypically, the Ws-eds1 mutant seedlings supported heavy sporulation by P. parasitica isolates that are each diagnostic for one of the RPP genes in wild-type Ws-0; none of the isolates is capable of sporulating on wild-type Ws-0. Ws-eds1 seedlings exhibited enhanced susceptibility to some P. parasitica isolates when compared with a compatible wild-type ecotype, Columbia, and the eds1 parental ecotype, Ws-0. This was observed as earlier initiation of sporulation and elevated production of conidiosporangia. Surprisingly, cotyledons of Ws-eds1 also supported low sporulation by five isolates of P. parasitica from Brassica oleracea. These isolates were unable to sporulate on > 100 ecotypes of Arabidopsis, including wild-type Ws-0. An isolate of Albugo candida (white blister) from B. oleracea also sporulated on Ws-eds1, but the mutant exhibited no alteration in phenotype when inoculated with several oomycete isolates from other host species. The bacterial resistance gene RPM1, conferring specific recognition of the avirulence gene avrB from Pseudomonas syringae pv glycinea, was not compromised in Ws-eds1 plants. The mutant also retained full responsiveness to the chemical inducer of systemic acquired resistance, 2,6-dichloroisonicotinic acid; Ws-eds1 seedlings treated with 2,6-dichloroisonicotinic acid became resistant to the Ws-0-compatible and Ws-0-incompatible P. parasitica isolates Emwa1 and Noco2, respectively. In summary, the EDS1 gene appears to be a necessary component of the resistance response specified by several RPP genes and is likely to function upstream from the convergence of disease resistance pathways in Arabidopsis.     

The downy mildew effector proteins ATR1 and ATR13 promote disease susceptibility in Arabidopsis thaliana

The downy mildew (Hyaloperonospora parasitica) effector proteins ATR1 and ATR13 trigger RPP1-Nd/WsB- and RPP13-Nd-dependent resistance, respectively, in Arabidopsis thaliana. To better understand the functions of these effectors during compatible and incompatible interactions of H. parasitica isolates on Arabidopsis accessions, we developed a novel delivery system using Pseudomonas syringae type III secretion via fusions of ATRs to the N terminus of the P. syringae effector protein, AvrRPS4. ATR1 and ATR13 both triggered the hypersensitive response (HR) and resistance to bacterial pathogens in Arabidopsis carrying RPP1-Nd/WsB or RPP13-Nd, respectively, when delivered from P. syringae pv tomato (Pst) DC3000. In addition, multiple alleles of ATR1 and ATR13 confer enhanced virulence to Pst DC3000 on susceptible Arabidopsis accessions. We conclude that ATR1 and ATR13 positively contribute to pathogen virulence inside host cells. Two ATR13 alleles suppressed bacterial PAMP (for Pathogen-Associated Molecular Patterns)-triggered callose deposition in susceptible Arabidopsis when delivered by DC3000 DeltaCEL mutants. Furthermore, expression of another allele of ATR13 in plant cells suppressed PAMP-triggered reactive oxygen species production in addition to callose deposition. Intriguingly, although Wassilewskija (Ws-0) is highly susceptible to H. parasitica isolate Emco5, ATR13Emco5 when delivered by Pst DC3000 triggered localized immunity, including HR, on Ws-0. We suggest that an additional H. parasitica Emco5 effector might suppress ATR13-triggered immunity.     

Arabidopsis RPP4 is a member of the RPP5 multigene family of TIR-NB-LRR genes and confers downy mildew resistance through multiple signalling components

In Arabidopsis, RPP4 confers resistance to Peronospora parasitica (P.p.) races Emoy2 and Emwa1 (downy mildew). We identified RPP4 in Col-0 as a member of the clustered RPP5 multigene family encoding nucleotide-binding leucine-rich repeat proteins with Toll/interleukin-1 receptor domains. RPP4 is the orthologue of RPP5 which, in addition to recognizing P.p. race Noco2, also mediates resistance to Emoy2 and Emwa1. Most differences between RPP4 and RPP5 occur in residues that constitute the TIR domain and in LRR residues that are predicted to confer recognition specificity. RPP4 requires the action of at least 12 defence components, including DTH9, EDS1, PAD4, PAL, PBS2, PBS3, SID1, SID2 and salicylic acid. The ndr1, npr1 and rps5-1 mutations partially compromise RPP4 function in cotyledons but not in true leaves. The identification of RPP4 as a TIR-NB-LRR protein, coupled with its dependence on certain signalling components in true leaves, is consistent with the hypothesis that distinct NB-LRR protein classes differentially signal through EDS1 and NDR1. Our results suggest that RPP4-mediated resistance is developmentally regulated and that in cotyledons there is cross-talk between EDS1 and NDR1 signalling and processes regulating systemic acquired resistance.     

Pronounced intraspecific haplotype divergence at the RPP5 complex disease resistance locus of Arabidopsis

In Arabidopsis ecotype Landsberg erecta (Ler), RPP5 confers resistance to the pathogen Peronospora parasitica. RPP5 is part of a clustered multigene family encoding nucleotide binding-leucine-rich repeat (LRR) proteins. We compared 95 kb of DNA sequence carrying the Ler RPP5 haplotype with the corresponding 90 kb of Arabidopsis ecotype Columbia (Col-0). Relative to the remainder of the genome, the Ler and Col-0 RPP5 haplotypes exhibit remarkable intraspecific polymorphism. The RPP5 gene family probably evolved by extensive recombination between LRRs from an RPP5-like progenitor that carried only eight LRRs. Most members have variable LRR configurations and encode different numbers of LRRs. Although many members carry retroelement insertions or frameshift mutations, codon usage analysis suggests that regions of the genes have been subject to purifying or diversifying selection, indicating that these genes were, or are, functional. The RPP5 haplotypes thus carry dynamic gene clusters with the potential to adapt rapidly to novel pathogen variants by gene duplication and modification of recognition capacity. We propose that the extremely high level of polymorphism at this complex resistance locus is maintained by frequency-dependent selection.     

A collection of INDEL markers for map-based cloning in seven Arabidopsis accessions

The availability of a comprehensive set of resources including an entire annotated reference genome, sequenced alternative accessions, and a multitude of marker systems makes Arabidopsis thaliana an ideal platform for genetic mapping. PCR markers based on INsertions/DELetions (INDELs) are currently the most frequently used polymorphisms. For the most commonly used mapping combination, Columbia×Landsberg erecta (Col-0×Ler-0), the Cereon polymorphism database is a valuable resource for the generation of polymorphic markers. However, because the number of markers available in public databases for accessions other than Col-0 and Ler-0 is extremely low, mapping using other accessions is far from straightforward. This issue arose while cloning mutations in the Wassilewskija (Ws-4) background. In this work, approaches are described for marker generation in Ws-4 x Col-0. Complementary strategies were employed to generate 229 INDEL markers. Firstly, existing Col-0/Ler-0 Cereon predicted polymorphisms were mined for transferability to Ws-4. Secondly, Ws-0 ecotype Illumina sequence data were analyzed to identify INDELs that could be used for the development of PCR-based markers for Col-0 and Ws-4. Finally, shotgun sequencing allowed the identification of INDELs directly between Col-0 and Ws-4. The polymorphism of the 229 markers was assessed in seven widely used Arabidopsis accessions, and PCR markers that allow a clear distinction between the diverged Ws-0 and Ws-4 accessions are detailed. The utility of the markers was demonstrated by mapping more than 35 mutations in a Col-0×Ws-4 combination, an example of which is presented here. The potential contribution of next generation sequencing technologies to more traditional map-based cloning is discussed.     

Cooperative ethylene and jasmonic acid signaling regulates selenite resistance in Arabidopsis

Selenium (Se) is an essential element for many organisms, but excess Se is toxic. To better understand plant Se toxicity and resistance mechanisms, we compared the physiological and molecular responses of two Arabidopsis (Arabidopsis thaliana) accessions, Columbia (Col)-0 and Wassilewskija (Ws)-2, to selenite treatment. Measurement of root length Se tolerance index demonstrated a clear difference between selenite-resistant Col-0 and selenite-sensitive Ws-2. Macroarray analysis showed more pronounced selenite-induced increases in mRNA levels of ethylene- or jasmonic acid (JA)-biosynthesis and -inducible genes in Col-0 than in Ws-2. Indeed, Col-0 exhibited higher levels of ethylene and JA. The selenite-sensitive phenotype of Ws-2 was attenuated by treatment with ethylene precursor or methyl jasmonate (MeJA). Conversely, the selenite resistance of Col-0 was reduced in mutants impaired in ethylene or JA biosynthesis or signaling. Genes encoding sulfur (S) transporters and S assimilation enzymes were up-regulated by selenite in Col-0 but not Ws-2. Accordingly, Col-0 contained higher levels of total S and Se and of nonprotein thiols than Ws-2. Glutathione redox status was reduced by selenite in Ws-2 but not in Col-0. Furthermore, the generation of reactive oxygen species by selenite was higher in Col-0 than in Ws-2. Together, these results indicate that JA and ethylene play important roles in Se resistance in Arabidopsis. Reactive oxygen species may also have a signaling role, and the resistance mechanism appears to involve enhanced S uptake and reduction.     

RCY1, an Arabidopsis thaliana RPP8/HRT family resistance gene,conferring resistance to cucumber mosaic virus requires salicylic acid,ethylene and a novel signal transduction mechanism

The dominant locus, RCY1, in the Arabidopsis thaliana ecotype C24 confers resistance to the yellow strain of cucumber mosaic virus (CMV-Y). The RCY1 locus was mapped to a 150-kb region on chromosome 5. Sequence comparison of this region from C24 and a CMV-Y-susceptible C24 mutant predicts that the RCY1 gene encodes a 104-kDa CC-NBS-LRR-type protein. The RCY1 gene from C24, when expressed in the susceptible ecotype Wassilewskija (Ws), restricted the systemic spread of virus. RCY1 is allelic to the resistance genes RPP8 from the ecotype Landsberg erecta and HRT from the ecotype Dijon-17, which confer resistance to Peronospora parasitica biotype Emco5 and turnip crinkle virus (TCV), respectively. Examination of RCY1 plants defective in salicylic acid (SA), jasmonic acid (JA) and ethylene signaling revealed a requirement for SA and ethylene signaling in mounting a resistance response to CMV-Y. The RCY1 nahG etr1 double mutants exhibited an intermediate level of susceptibility to CMV-Y, compared to the resistant ecotype C24 and the susceptible ecotypes Columbia and Nossen. This suggests that in addition to SA and ethylene, a novel signaling mechanism is associated with the induction of resistance in CMV-Y-infected C24 plants. Moreover, our results suggest that the signaling pathways downstream of the RPP8, HRT, and RCY1 have evolved independently.     

Phenotypic characterization and molecular mapping of the Arabidopsis thaliana locus RPP5, determining disease resistance to Peronospora parasitica

Peronospora parasitica causes downy mildew on crucifers. An isolate of P. parasitica (denoted NoCO2) was identified that infected Arabidopsis plants of the land race Columbia (Col-0) but not plants of land race Landsberg erecta (La- er ). Segregation analysis of F₂ plants derived from a La- er x Col-0 cross established that the resistance was inherited as a single locus, denoted RPP5 . Macroscopic and microscopic examinations of inoculated La- er and Col-0 cotyledons showed that restriction of fungal growth in La- er was accompanied by massive callose accumulation and death of plant cells in direct contact with points of attempted fungal penetration. La- er x Col-0 F₁ plants exhibited an intermediate resistance response in all aspects of fungal development, indicating that RPP5 is semi-dominant in its action. F₈ recombinant inbred lines generated between La- er and Col-0 were used to map RPP5 to a narrow interval (<1.1 cM) on chromosome 4, utilizing existing restriction fragment length polymorphic (RFLP) markers and newly generated random amplified polymorphic DNA (RAPD) markers. The data provide a basis for the isolation of the RPP5 locus by positional cloning as a first step towards understanding recognitional specificity in plant-pathogen interactions at a molecular level.     

The Arabidopsis thaliana TIR-NB-LRR R-protein, RPP1A; protein localization and constitutive activation of defence by truncated alleles in tobacco and Arabidopsis

Specific recognition of Hyaloperonospora parasitica isolate Cala2 by Arabidopsis thaliana Ws-0 is mediated by the resistance gene RPP1A. Transient expression of different truncations of RPP1A in tobacco leaves revealed that its TIR-NB-ARC portion is sufficient to induce an elicitor-independent cell death. In stable transgenic lines of Arabidopsis, overexpression of the RPP1A TIR-NB-ARC domains (E12) using the 35S promoter leads to broad-spectrum resistance to virulent strains of H. parasitica and Pseudomonas syringae DC3000. The TIR-NB-ARC-mediated constitutive immunity is due to activation of the salicylic acid-dependent resistance pathway and is relieved by either a mutation in EDS1 or the presence of the salicylate hydroxylase gene, NahG. Growth of 35S::E12 plants is reduced, a phenotype observed in many constitutively resistant mutants. RPP1A carries a hydrophobic peptide at its N-terminus that directs the RPP1A protein into membranes, though it may not be the sole determinant mediating membrane association of RPP1A. Two-phase partitioning and sucrose density gradient sedimentation established that RPP1A resides in the endoplasmic reticulum and/or Golgi apparatus.     

Plant growth-promoting rhizobacteria systemically protect Arabidopsis thaliana against Cucumber mosaic virus by a salicylic acid and NPR1-independent and jasmonic acid-dependent signaling pathway

Arabidopsis thaliana ecotype Columbia plants (Col-0) treated with plant growth-promoting rhizobacteria (PGPR) Serattia marcescens strain 90-166 and Bacillus pumilus strain SE34 had significantly reduced symptom severity by Cucumber mosaic virus (CMV). In some cases, CMV accumulation was also significantly reduced in systemically infected leaves. The signal transduction pathway(s) associated with induced resistance against CMV by strain 90-166 was determined using mutant strains and transgenic and mutant Arabidopsis lines. NahG plants treated with strains 90-166 and SE34 had reduced symptom severity indicating that the resistance did not require salicylic acid (SA). Strain 90-166 naturally produces SA under iron-limited conditions. Col-0 and NahG plants treated with the SA-deficient mutant, 90-166-1441, had significantly reduced CMV symptom severity with reduced virus accumulation in Col-0 plants. Another PGPR mutant, 90-166-2882, caused reduced disease severity in Col-0 and NahG plants. In a time course study, strain 90-166 reduced virus accumulation at 7 but not at 14 and 21 days post-inoculation (dpi) on the non-inoculated leaves of Col-0 plants. NahG and npr1-1 plants treated with strain 90-166 had reduced amounts of virus at 7 and 14 dpi but not at 21 dpi. In contrast, no decrease in CMV accumulation occurred in strain 90-166-treated fad3-2 fad7-2 fad8 plants. These data indicate that the protection of Arabidopsis against CMV by strain 90-166 follows a signaling pathway for virus protection that is independent of SA and NPR1, but dependent on jasmonic acid.     

Transgressive segregation reveals two Arabidopsis TIR-NB-LRR resistance genes effective against Leptosphaeria maculans, causal agent of blackleg disease

In a cross between the two resistant accessions Col-0 and Ler-0, a 15:1 segregation was found in F2, suggesting the presence of unlinked resistance loci to Leptosphaeria maculans. One hundred Col-4 x Ler-0, and 50 Ler-2 x Cvi-1 recombinant inbred lines, and seven susceptible Ler-0 x Ws-0 F2 progenies were examined to identify the two loci. Resistance in Col-4, Ws-0 and Cvi-1 (RLM1) was mapped to the marker m305 on chromosome 1. Col-4 x Ler-0 and Ler-2 x Cvi-1 mapping populations located RLM2(Ler) on the same arm of chromosome 4. A tight physical location of RLM2 was established through near-isogenic lines. This region was found to correspond to an ancient duplication event between the RLM1 and RLM2 loci. Two independent T-DNA mutants in a TIR-NB-LRR R gene (At1g64070) displayed susceptibility, and L. maculans susceptible mutant phenotypes were confirmed to be allelic for rlm1 in F1 after crosses with susceptible rlm1(Ler)rlm2(Col) plants. Complementation of rlm1(Ler)rlm2(Col) with the genomic Col-0 sequence of At1g64070 conferred resistance. In addition, two T-DNA mutants in a neighbouring homologous TIR-NB-LRR gene (At1g63880) displayed moderate susceptibility to L. maculans. Sequence analysis revealed that At1g64070 was truncated by a premature stop codon, and that At1g63880 was absent in Ler-0. RNA interference confirmed that Ler-0 resistance is dependent on genes structurally related to RLM1. Camalexin was identified as a quantitative co-dominant resistance factor of Col-0 origin, but independent of RLM1. RLM1/RLM2 resistance was, however, found to require RAR1 and partially HSP90.1.     

A single locus determines sensitivity to bacterial flagellin in Arabidopsis thaliana

Peptides corresponding to the most conserved domain of eubacterial flagellin act as potent elicitors in cells of different plant species. In intact Arabidposis thaliana seedlings these peptides (flg22 and flg15) caused callose deposition, induction of genes coding for pathogenesis-related proteins and a strong inhibition of growth. Half-maximal growth inhibition occurred at peptide concentrations of approximately 100 nM. In contrast, peptides representing the corresponding flagellin domains of the plant-associated bacteria A. tumefaciens and R. meliloti were inactive even at concentrations of 10 microM. With the exception of Ws-0, all ecotypes of A. thaliana tested were sensitive to flg22. Crosses of Ws-0 with the sensitive ecotypes Col-0 and La-er, respectively, resulted in sensitive F1 seedlings. In the F2 generation of both crosses, sensitivity segregated as a single trait with markers of chromosome 5 and a ratio of 3:1. Dominance of the locus sensing flagellin, termed FLS-1, suggests that it encodes an element which is important for the perception of the flagellin signal.     

The floral transition is not the developmental switch that confers competence for the Arabidopsis age-related resistance response to Pseudomonas syringae pv. tomato

Age-related resistance (ARR) is a plant defense response characterized by enhanced resistance to certain pathogens in mature plants relative to young plants. In Arabidopsis thaliana the transition to flowering is associated with ARR competence, suggesting that this developmental event is the switch that initiates ARR competence in mature plants (Rusterucci et al. in Physiol Mol Plant Pathol 66:222–231, 2005). The association of ARR and the floral transition was examined using flowering-time mutants and photoperiod-induced flowering to separate flowering from other developmental events that occur as plants age. Under short-day conditions, late-flowering plant lines ld-1 (luminidependens-1), soc1-2 (suppressor of overexpression of co 1-2), and FRI ⁺ (FRIGIDA) displayed ARR before the transition to flowering occurred. Early-flowering svp-31, svp-32 (short vegetative phase), and Ws-2 were ARR-defective, whereas early-flowering tfl1-14 (terminal flower 1-14) displayed ARR at the same time as Col-0. While svp-31, svp-32 and Ws-2 produced few rosette leaves, tfl1-14 produced a rosette leaf number similar to Col-0, suggesting that the development of a minimum number of rosette leaves is necessary to initiate ARR competence under short-day conditions. Photoperiod-induced transient expression of FT (FLOWERING LOCUS T) caused precocious flowering in short-day-grown Col-0 but this was not associated with ARR competence. Under long-day conditions co-9 (constans-9) mutants did not flower but displayed an ARR response at the same time as Col-0. This study suggests that SVP is required for the ARR response and that the floral transition is not the developmental event that regulates ARR competence.     

Somatic embryogenesis in cultured immature zygotic embryos and leaf protoplasts of Arabidopsis thaliana ecotypes

Immature zygotic embryos of six ecotypes (Nd-0, Ler, C24, Col-0, Nossen, Ws-2) of Arabidopsis thaliana (L.) Heynh. were cultured in vitro. The same ecotypes, except Nossen, were used for studies on leaf protoplast culture. Experimental conditions for the induction of somatic embryos were established in both culture systems. In the case of immature zygotic embryos, the parameters investigated were the influence of developmental stage of the explant, the ecotypes used, and various concentrations and combinations of growth regulatory substances (phytohormones). In the ecotype Ler, structures were discovered which were very similar to those found in the early stages of zygotic embryogenesis: globular structures at the end of a suspensor-like single file of cells were frequently observed. In the case of leaf protoplasts, high efficiencies of colony formation and plant regeneration occurred in Ws-2 and C24. A novel type of cell division pattern was found in Col-0 and C24, again highly reminiscent of the early division patterns in zygotic embryos. Similarities and differences between zygotic and somatic embryogenesis are discussed. Received: 2 August 1996 / Accepted: 4 February 1997     

机械损伤对拟南芥莲座叶芥子油苷含量和组成的影响

植物可以利用体内次生代谢产物的变化来抵御昆虫取食和机械损伤.芥子油苷是拟南芥的主要次生代谢产物.通过剪刀剪取叶片(40%面积)对温室培养的拟南芥幼苗莲座叶进行机械损伤处理,观察机械损伤后8个时间点拟南芥叶片中不同种类芥子油苷含量和组合模式的变化.结果表明机械损伤后3 h叶片中芥子油苷总含量开始明显上升,脂肪族和吲哚族芥子油苷含量在损伤后3 h也都显著高于损伤前.在检测到的12种芥子油苷中,4-甲基亚磺酰丁基芥子油苷(4-methylsulphinylbutyl GS,4MSOB)的含量最多,占芥子油苷总量的48.5%,并且在损伤3 h后含量增加.4MSOB含量的变化成为影响莲座叶中芥子油苷组合模式的主导因素.其它各种芥子油苷在损伤后不同时间点的变化也存在差异.     

Genetic and physical mapping of the RPP13 locus, in Arabidopsis, responsible for specific recognition of several Peronospora parasitica (downy mildew) isolates.

Fifteen isolates of the biotrophic oomycete Peronospora parasitica (downy mildew) were obtained from a population of Arabidopsis thaliana plants that established naturally in a garden the previous year. They exhibited phenotypic variation in a set of 12 Arabidopsis accessions that suggested that the parasite population consisted of at least six pathotypes. One isolate, Maks9, elicited an interaction phenotype of flecking necrosis and no sporulation (FN) in the Arabidopsis accession Nd-1, and more extensive pitting necrosis with no sporulation (PN) in the accession Ws-2. RPP13 was designated as the locus for a single dominant resistance gene associated with the resistance in Nd-1 and mapped to an interval of approximately 60 kb on a bacterial artificial chromosome (BAC) contig on the lower arm of chromosome 3. This locus is approximately 6 cM telomeric to RPP1, which was previously described as the locus for the PN interaction with five Peronospora isolates, including resistance to Maks9 in Ws-2. New Peronospora isolates were obtained from four other geographically distinct populations of P. parasitica. Four isolates were characterized that elicited an FN phenotype in Nd-1 and mapped resistance to the RPP13 locus. This suggests that the RPP13 locus contains either a single gene capable of multiple isolate recognition or a group of tightly linked genes. Further analysis suggests that the RPP11 gene in the accession Rld-0 may be allelic to RPP13 but results in a different recognition capability.