Characterization of Arabidopsis mur3 mutations that result in constitutive activation of defence in petioles, but not leaves

A screen was established for mutants in which the plant defence response is de-repressed. The pathogen-inducible isochorismate synthase (ICS1) promoter was fused to firefly luciferase (luc) and a homozygous transgenic line generated in which the ICS1:luc fusion is co-regulated with ICS1. This line was mutagenized and M(2) seedlings screened for constitutive ICS1:luc expression (cie). The cie mutants fall into distinct phenotypic classes based on tissue-specific localization of luciferase activity. One mutant, cie1, that shows constitutive luciferase activity specifically in petioles, was chosen for further analysis. In addition to ICS1, PR and other defence-related genes are constitutively expressed in cie1 plants. The cie1 mutant is also characterized by an increased production of conjugated salicylic acid and reactive oxygen intermediates, as well as spontaneous lesion formation, all confined to petiole tissue. Significantly, defences activated in cie1 are sufficient to prevent infection by a virulent isolate of Hyaloperonospora parasitica, and this enhanced resistance response protects petiole tissue alone. Furthermore, cie1-mediated resistance, along with PR gene expression, is abolished in a sid2-1 mutant background, consistent with a requirement for salicylic acid. A positional cloning approach was used to identify cie1, which carries two point mutations in a gene required for cell wall biosynthesis and actin organization, MUR3. A mur3 knockout mutant also resists infection by H. parasitica in its petioles and this phenotype is complemented by transformation with wild-type MUR3. We propose that perturbed cell wall biosynthesis may activate plant defence and provide a rationale for the cie1 and the mur3 knockout phenotypes.     

Herbivore-induced resistance against microbial pathogens in Arabidopsis

Caterpillars of the herbivore Pieris rapae stimulate the production of jasmonic acid (JA) and ethylene (ET) in Arabidopsis (Arabidopsis thaliana) and trigger a defense response that affects insect performance on systemic tissues. To investigate the spectrum of effectiveness of P. rapae-induced resistance, we examined the level of resistance against different pathogens. Although the necrotrophic fungus Alternaria brassicicola is sensitive to JA-dependent defenses, herbivore-induced resistance was not effective against this pathogen. By contrast, caterpillar feeding significantly reduced disease caused by the bacterial pathogens Pseudomonas syringae pv tomato and Xanthomonas campestris pv armoraciae. However, this effect was apparent only locally in caterpillar-damaged tissue. Arabidopsis mutants jar1, coi1, ein2, sid2, eds5, and npr1 showed wild-type levels of P. rapae-induced protection against P. syringae pv tomato, suggesting that this local, herbivore-induced defense response does not depend exclusively on either JA, ET, or salicylic acid (SA). Resistance against the biotroph Turnip crinkle virus (TCV) requires SA, but not JA and ET. Nevertheless, herbivore feeding strongly affected TCV multiplication and TCV lesion formation, also in systemic tissues. Wounding alone was not effective, but application of P. rapae regurgitate onto the wounds induced a similar level of protection. Analysis of SA-induced PATHOGENESIS RELATED-1 (PR-1) expression revealed that P. rapae grazing primed Arabidopsis leaves for augmented expression of SA-dependent defenses. Pharmacological experiments showed that ET acts synergistically on SA-induced PR-1, suggesting that the increased production of ET upon herbivore feeding sensitizes the tissue to respond faster to SA, thereby contributing to an enhanced defensive capacity toward pathogens, such as TCV, that trigger SA-dependent defenses upon infection.     

A role for a menthone reductase in resistance against microbial pathogens in plants

Plants elaborate a vast array of enzymes that synthesize defensive secondary metabolites in response to pathogen attack. Here, we isolated the pathogen-responsive CaMNR1 [menthone: (+)-(3S)-neomenthol reductase] gene, a member of the short-chain dehydrogenase/reductase (SDR) superfamily, from pepper (Capsicum annuum) plants. Gas chromatography-mass spectrometry analysis revealed that purified CaMNR1 and its ortholog AtSDR1 from Arabidopsis (Arabidopsis thaliana) catalyze a menthone reduction with reduced nicotinamide adenine dinucleotide phosphate as a cofactor to produce neomenthol with antimicrobial activity. CaMNR1 and AtSDR1 also possess a significant catalytic activity for neomenthol oxidation. We examined the cellular function of the CaMNR1 gene by virus-induced gene silencing and ectopic overexpression in pepper and Arabidopsis plants, respectively. CaMNR1-silenced pepper plants were significantly more susceptible to Xanthomonas campestris pv vesicatoria and Colletotrichum coccodes infection and expressed lower levels of salicylic acid-responsive CaBPR1 and CaPR10 and jasmonic acid-responsive CaDEF1. CaMNR1-overexpressing Arabidopsis plants exhibited enhanced resistance to the hemibiotrophic pathogen Pseudomonas syringae pv tomato DC3000 and the biotrophic pathogen Hyaloperonospora parasitica isolate Noco2, accompanied by the induction of AtPR1 and AtPDF1.2. In contrast, mutation in the CaMNR1 ortholog AtSDR1 significantly enhanced susceptibility to both pathogens. Together, these results indicate that the novel menthone reductase gene CaMNR1 and its ortholog AtSDR1 positively regulate plant defenses against a broad spectrum of pathogens.     

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.     

Screening for resistance against Pseudomonas syringae in rice-FOX Arabidopsis lines identified a putative receptor-like cytoplasmic kinase gene that confers resistance to major bacterial and fungal pathogens in Arabidopsis and rice

Approximately 20,000 of the rice-FOX Arabidopsis transgenic lines, which overexpress 13,000 rice full-length cDNAs at random in Arabidopsis, were screened for bacterial disease resistance by dip inoculation with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The identities of the overexpressed genes were determined in 72 lines that showed consistent resistance after three independent screens. Pst DC3000 resistance was verified for 19 genes by characterizing other independent Arabidopsis lines for the same genes in the original rice-FOX hunting population or obtained by reintroducing the genes into ecotype Columbia by floral dip transformation. Thirteen lines of these 72 selections were also resistant to the fungal pathogen Colletotrichum higginsianum. Eight genes that conferred resistance to Pst DC3000 in Arabidopsis have been introduced into rice for overexpression, and transformants were evaluated for resistance to the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. One of the transgenic rice lines was highly resistant to Xanthomonas oryzae pv. oryzae. Interestingly, this line also showed remarkably high resistance to Magnaporthe grisea, the fungal pathogen causing rice blast, which is the most devastating rice disease in many countries. The causal rice gene, encoding a putative receptor-like cytoplasmic kinase, was therefore designated as BROAD-SPECTRUM RESISTANCE 1. Our results demonstrate the utility of the rice-FOX Arabidopsis lines as a tool for the identification of genes involved in plant defence and suggest the presence of a defence mechanism common between monocots and dicots.     

Parasite-host fitness trade-offs change with parasite identity: genotype-specific interactions in a plant-pathogen system

Simultaneous effects of host and parasite in determining quantitative traits of infection have long been neglected in theoretical and experimental investigations of host-parasite coevolution with the notable exception of gene-for-gene resistance studies. A cross-infection experiment, using five lines of the plant Arabidopsis thaliana and two strains of its oomycete pathogen Hyaloperonospora parasitica, revealed that three traits traditionally considered those of the parasite (number of infected leaves, transmission success, and time until 50% transmission), differed among specific combinations of host and parasite lines, being determined by the two protagonists of the infection. However, the two parasite strains did not differ significantly for most measured phenotypic traits of the infection. Globally, transmission increased with increasing virulence among the different host-parasite combinations, as assumed by most models of evolution of virulence. Surprisingly, however, there was no general relationship between parasite and host fitness, estimated respectively as transmission and seed production. Only one of the two strains showed the expected significant negative genetic correlation between these two variables. Our results thus highlight the importance of taking into account both host and parasite genetic variation because their interaction can lead to unexpected evolutionary outcomes.     

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.     

A critical role for disproportionating enzyme in starch breakdown is revealed by a knock-out mutation in Arabidopsis

Disproportionating enzyme (D-enzyme) is a plastidial alpha-1,4-glucanotransferase but its role in starch metabolism is unclear. Using a reverse genetics approach we have isolated a mutant of Arabidopsis thaliana in which the gene encoding this enzyme (DPE1) is disrupted by a T-DNA insertion. While D-enzyme activity is eliminated in the homozygous dpe1-1 mutant, changes in activities of other enzymes of starch metabolism are relatively small. During the diurnal cycle, the amount of leaf starch is higher in dpe1-1 than in wild type and the amylose to amylopectin ratio is increased, but amylopectin structure is unaltered. The amounts of starch synthesised and degraded are lower in dpe1-1 than in wild type. However, the lower amount of starch synthesised and the higher proportion of amylose are both eliminated when plants are completely de-starched by a period of prolonged darkness prior to the light period. During starch degradation, a large accumulation of malto-oligosaccharides occurs in dpe1-1 but not in wild type. These data show that D-enzyme is required for malto-oligosaccharide metabolism during starch degradation. The slower rate of starch degradation in dpe1-1 suggests that malto-oligosaccharides affect an enzyme that attacks the starch granule, or that D-enzyme itself can act directly on starch. The effects on starch synthesis and composition in dpe1-1 under normal diurnal conditions are probably a consequence of metabolism at the start of the light period, of the high levels of malto-oligosaccharides generated during the dark period. We conclude that the primary function of D-enzyme is in starch degradation.     

A high-throughput chemical screen for resistance to Pseudomonas syringae in Arabidopsis

The study of plant pathogenesis and the development of effective treatments to protect plants from diseases could be greatly facilitated by a high-throughput pathosystem to evaluate small-molecule libraries for inhibitors of pathogen virulence. The interaction between the Gram-negative bacterium Pseudomonas syringae and Arabidopsis thaliana is a model for plant pathogenesis. However, a robust high-throughput assay to score the outcome of this interaction is currently lacking. We demonstrate that Arabidopsis seedlings incubated with P. syringae in liquid culture display a macroscopically visible 'bleaching' symptom within 5 days of infection. Bleaching is associated with a loss of chlorophyll from cotyledonary tissues, and is correlated with bacterial virulence. Gene-for-gene resistance is absent in the liquid environment, possibly because of the suppression of the hypersensitive response under these conditions. Importantly, bleaching can be prevented by treating seedlings with known inducers of plant defence, such as salicylic acid (SA) or a basal defence-inducing peptide of bacterial flagellin (flg22) prior to inoculation. Based on these observations, we have devised a high-throughput liquid assay using standard 96-well plates to investigate the P. syringae-Arabidopsis interaction. An initial screen of small molecules active on Arabidopsis revealed a family of sulfanilamide compounds that afford protection against the bleaching symptom. The most active compound, sulfamethoxazole, also reduced in planta bacterial growth when applied to mature soil-grown plants. The whole-organism liquid assay provides a novel approach to probe chemical libraries in a high-throughput manner for compounds that reduce bacterial virulence in plants.     

A PCR assay for the quantification of growth of the oomycete pathogen Hyaloperonospora arabidopsidis in Arabidopsis thaliana

The accurate quantification of disease severity is important for the assessment of host–pathogen interactions in laboratory or field settings. The interaction between Arabidopsis thaliana and its naturally occurring downy mildew pathogen, Hyaloperonospora arabidopsidis (Hpa), is a widely used reference pathosystem for plant–oomycete interactions. Current methods for the assessment of disease severity in the Arabidopsis–Hpa interaction rely on measurements at the terminal stage of pathogen development; namely, visual counts of spore‐producing structures or the quantification of spore production with a haemocytometer. These assays are useful, but do not offer sensitivity for the robust quantification of small changes in virulence or the accurate quantification of pathogen growth prior to the reproductive stage. Here, we describe a quantitative real‐time polymerase chain reaction (qPCR) assay for the monitoring of Hpa growth in planta. The protocol is rapid, inexpensive and can robustly distinguish small changes in virulence. We used this assay to investigate the dynamics of early Hpa mycelial growth and to demonstrate the proof of concept that this assay could be used in screens for novel oomycete growth inhibitors.     

Structure‐function analysis of npr1 alleles in Arabidopsis reveals a role for its paralogs in the perception of salicylic acid

Salicylic acid (SA) is necessary for plant defence against some pathogens, whereas NPR1 is necessary for SA perception. Plant defence can be induced to an extreme by several applications of benzothiadiazole (BTH), an analogue of SA. Thus, plants that do not perceive BTH grow unaffected, whereas wild‐type plants grow stunted. This feature allows us to screen for mutants in Arabidopsis thaliana that show insensitivity to BTH in a high‐throughput fashion. Most of the mutants are npr1 alleles, with similar phenotypes in plant weight and pathogen growth. The mutations are clustered in the carboxyl‐terminal part of the protein, and no obvious null alleles were recovered. These facts have prompted a search for knockouts in the NPR1 gene. Two of these KO alleles identified are null and have an intermediate phenotype. All the evidence presented lead us to propose a redundancy in SA perception, with the paralogs of NPR1 taking part in this signalling. We show that the mutations recovered in the screening genetically interact with the paralogs preventing their function in SA signalling.     

一种筛选拟南芥突变体的有效方法

经甲基磺酸乙酯（EMS）诱变处理的拟南芥种子,接种于MS培养基上,垂直放置培养4天后,将幼苗转移至胁迫培养基中,以倒置幼苗180°所形成的弯曲生长根作为指标筛选拟南芥耐营养胁迫突变体。利用这种方法,成功地筛选到一个耐低钾的隐性单基因拟南芥突变体。本方法同样适用于其他类型突变体的筛选。 Abstract：his paper introduces a root-bending assay for isol ation of Arabidopsis mutants tolerant to nutrition stress. Seeds of wild-ty pe Arabidopsis thaliana (ecotype Landersberg erecta) were mutagenized wi th ethyl methyl sulfide (EMS),and M2 populations were screened for mutants. Fo ur-day-old seedlings with 1-to 1.5-cm-long roots were transferred from the vertical agar plates onto to a second agar medium that was supplemented with det erminate stress. The seedlings were arranged in rows, and the plates were orient ed vertically with the roots pointing upward. After another 4 days, the root be nding seedlings were selected for putative mutants and transferred to soil to gr ow to maturity.Seeds from the putative mutants were screened again to determine the true mutants.By using this root-bending assay we have isolated a low-K+-tolerant (lkt1) mutant which is caused by single recessive nuclear mutation. F or lkt1 mutant screening,K+concentration of the medium was 100μmol/L because root growth of wild type seedlings was completely inhibited at or below this con centration.This root-bending assay is also applicable to other type of Arabid opsis mutant isolation.     

Study of the role of antimicrobial glucosinolate-derived isothiocyanates in resistance of Arabidopsis to microbial pathogens

Crude aqueous extracts from Arabidopsis leaves were subjected to chromatographic separations, after which the different fractions were monitored for antimicrobial activity using the fungus Neurospora crassa as a test organism. Two major fractions were obtained that appeared to have the same abundance in leaves from untreated plants versus leaves from plants challenge inoculated with the fungus Alternaria brassicicola. One of both major antimicrobial fractions was purified to homogeneity and identified by 1H nuclear magnetic resonance, gas chromatography/electron impact mass spectrometry, and gas chromatography/chemical ionization mass spectrometry as 4-methylsulphinylbutyl isothiocyanate (ITC). This compound has previously been described as a product of myrosinase-mediated breakdown of glucoraphanin, the predominant glucosinolate in Arabidopsis leaves. 4-Methylsulphinylbutyl ITC was found to be inhibitory to a wide range of fungi and bacteria, producing 50% growth inhibition in vitro at concentrations of 28 microM for the most sensitive organism tested (Pseudomonas syringae). A previously identified glucosinolate biosynthesis mutant, gsm1-1, was found to be largely deficient in either of the two major antimicrobial compounds, including 4-methylsulphinylbutyl ITC. The resistance of gsm1-1 was compared with that of wild-type plants after challenge with the fungi A. brassicicola, Plectosphaerella cucumerina, Botrytis cinerea, Fusarium oxysporum, or Peronospora parasitica, or the bacteria Erwinia carotovora or P. syringae. Of the tested pathogens, only F. oxysporum was found to be significantly more aggressive on gsm1-1 than on wild-type plants. Taken together, our data suggest that glucosinolate-derived antimicrobial ITCs can play a role in the protection of Arabidopsis against particular pathogens.