病毒诱导的基因沉默及其在植物基因功能研究中的应用

RNA介导的基因沉默是近年来在生物体中发现的一种基于核酸水平高度保守的特异性降解机制.病毒诱导的基因沉默（virus induced gene silencing, VIGS）是指携带植物功能基因cDNA的病毒在侵染植物体后,可诱导植物发生基因沉默而出现表型突变,进而可以研究该目的基因功能.至今,已经建立了以RNA病毒、DNA病毒、卫星病毒和DNA卫星分子为载体的VIGS体系,这些病毒载体能在多种寄主植物（包括拟南芥、番茄和大麦）上有效抑制功能基因的表达.VIGS已开始应用于N基因和Pto基因介导的抗性信号途径中关键基因的功能研究、抗病毒相关的寄主因子研究以及植物代谢和发育调控研究.在当前植物基因组或EST序列大量测定的情况下,VIGS为植物基因功能鉴定提供了有效的技术平台.     

The MI-1-mediated pest resistance requires Hsp90 and Sgt1

The tomato (Solanum lycopersicum) Mi-1 gene encodes a protein with putative coiled-coil nucleotide-binding site and leucine-rich repeat motifs. Mi-1 confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphids (Macrosiphum euphorbiae), and sweet potato whitefly (Bemisia tabaci). To identify genes required in the Mi-1-mediated resistance to nematodes and aphids, we used tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) to repress candidate genes and assay for nematode and aphid resistance. We targeted Sgt1 (suppressor of G-two allele of Skp1), Rar1 (required for Mla12 resistance), and Hsp90 (heat shock protein 90), which are known to participate early in resistance gene signaling pathways. Two Arabidopsis (Arabidopsis thaliana) Sgt1 genes exist and one has been implicated in disease resistance. Thus far the sequence of only one Sgt1 ortholog is known in tomato. To design gene-specific VIGS constructs, we cloned a second tomato Sgt1 gene, Sgt1-2. The gene-specific VIGS construct TRV-SlSgt1-1 resulted in lethality, while silencing Sgt1-2 using TRV-SlSgt1-2 did not result in lethal phenotype. Aphid and root-knot nematode assays of Sgt1-2-silenced plants indicated no role for Sgt1-2 in Mi-1-mediated resistance. A Nicotiana benthamiana Sgt1 VIGS construct silencing both Sgt1-1 and Sgt1-2 yielded live plants and identified a role for Sgt1 in Mi-1-mediated aphid resistance. Silencing of Rar1 did not affect Mi-1-mediated nematode and aphid resistance and demonstrated that Rar1 is not required for Mi-1 resistance. Silencing Hsp90-1 resulted in attenuation of Mi-1-mediated aphid and nematode resistance and indicated a role for Hsp90-1. The requirement for Sgt1 and Hsp90-1 in Mi-1-mediated resistance provides further evidence for common components in early resistance gene defense signaling against diverse pathogens and pests.     

病毒诱导基因沉默鉴定穿心莲内酯生物合成关键酶ApCPS功能

该研究采用病毒诱导基因沉默技术(VIGS),以生长到第8片真叶期的穿心莲植株为实验材料,沉默参与穿心莲内酯生物合成的ent-柯巴基焦磷酸合酶基因(ApCPS),用半定量和荧光定量PCR检测病毒诱导沉默后ApCPS及其上游基因的表达,用HPLC法检测ApCPS沉默后穿心莲内酯的积累变化,同时检测茉莉酸甲酯(MeJA)处理后ApCPS及上游基因的表达,以全面分析穿心莲内酯代谢以及ApCPS在穿心莲内酯生物合成中的作用机制,验证其在植物体内的功能。结果显示:(1)ApCPS基因被成功沉默,基因表达显著下调,进而引起上游牻牛儿基牻牛儿基焦磷酸合成酶基因(GGPS)的表达下调,而3-羟-3-甲基戊二酰辅酶A还原酶基因(HMGR)和1-脱氧木酮糖-5-磷酸合成酶基因(DXS)的表达未受影响。(2)ApCPS基因沉默15d后穿心莲内酯积累量显著下降,表明ApCPS是穿心莲内酯生物合成关键酶基因,且能够负反馈影响上游基因表达。(3)茉莉酸甲酯(MeJA)显著诱导ApCPS及上游基因HMGR、DXS和GGPS的表达,表明穿心莲内酯生物合成基因受到MeJA的广泛调控。该研究首次使用VIGS证明ApCPS参与到穿心莲内酯生物合成,为利用该技术鉴定穿心莲内酯生物合成途径中其他基因功能奠定了基础。     

Virus-induced silencing of WIPK and SIPK genes reduces resistance to a bacterial pathogen,but has no effect on the INF1-induced hypersensitive response (HR) in Nicotiana benthamiana

Activation of two mitogen-activated protein kinases (MAPKs), wound-induced protein kinase (WIPK) and salicylic acid-induced protein kinase (SIPK), is one of the earliest responses that occur in tobacco plants that have been wounded, treated with pathogen-derived elicitors or challenged with avirulent pathogens. We isolated cDNAs for these MAPKs ( NbWIPK and NbSIPK) from Nicotiana benthamiana. The function of NbWIPK and NbSIPK in mediating the hypersensitive response (HR) triggered by infiltration with INF1 protein (the major elicitin secreted by Phytophthora infestans), and the defense response to an incompatible bacterial pathogen ( Pseudomonas cichorii), was investigated by employing virus-induced gene silencing (VIGS) to inhibit expression of the WIPK and SIPK genes in N. benthamiana. Silencing of WIPK or SIPK, or both genes simultaneously, resulted in reduced resistance to P. cichorii, but no change was observed in the timing or extent of HR development after treatment with INF1.Communicated by R. G. Herrmann     

New disease resistance genes in soybean against Pseudomonas syringae pv glycinea: evidence that one of them interacts with a bacterial elicitor

Summary Soybean [Glycine max (L.) Merr.] cultivars Flambeau and Merit differed in their resistance to Pseudomonas syringae pv glycinea (Psg) race 4, carrying each of four different avirulence (avr) genes cloned from Psg or the related bacterium, Pseudomonas syringae pv tomato. Segregation data for F₂ and F₃ progeny of Flambeau x Merit crosses indicated that single dominant and nonallelic genes account for resistance to Psg race 4, carrying avirulence genes avrA, avrB, avrC, or avrD. Segregants were also recovered that carried all four or none of the disease resistance genes. One of the disease resistance genes (Rpg1, complementing bacterial avirulence gene B) had been described previously, but the other three genes — designated Rpg2, Rpg3, and Rpg4 — had not here to fore been defined. Rpg3 and Rpg4 are linked (40.5 ± 3.2 recombination units). Rpg4 complements avrD, cloned from Pseudomonas syringae pv tomato, but a functional copy of this avirulence gene has not thus far been observed in Pseudomonas syringae pv glycinea. Resistance gene Rpg4 therefore may account in part for the resistance of soybean to Pseudomonas syringae pv tomato and other pathogens harboring avrD.     

Glucocorticoid‐inducible expression of a bacterial avirulence gene in transgenic Arabidopsis induces hypersensitive cell death

Pathogenic strains of Pseudomonas syringae pv. tomato carrying the avrRpt2 avirulence gene specifically induce a hypersensitive cell death response in Arabidopsis plants that contain the complementary RPS2 disease resistance gene. Transient expression of avrRpt2 in Arabidopsis plants having the RPS2 gene has been shown to induce hypersensitive cell death. In order to analyze the effects of conditional expression of avrRpt2 in Arabidopsis plants, transgenic lines were constructed that contained the avrRpt2 gene under the control of a tightly regulated, glucocorticoid-inducible promoter. Dexamethasone-induced expression of avrRpt2 in transgenic lines having the RPS2 gene resulted in a specific hypersensitive cell death response that resembled a Pseudomonas syringae-induced hypersensitive response and also induced the expression of a pathogenesis-related gene (PR1). Interestingly, high level expression of avrRpt2 in a mutant rps2–101C background resulted in plant stress and ultimately cell death, suggesting a possible role for avrRpt2 in Pseudomonas syringae virulence. Transgenic RPS2 and rps2 plants that contain the glucocorticoid-inducible avrRpt2 gene will provide a powerful new tool for the genetic, physiological, biochemical, and molecular dissection of an avirulence gene-specified cell death response in both resistant and susceptible plants.     

Expression of the Hypersensitive Response-assisting Protein in <Emphasis Type="Italic">Arabidopsis</Emphasis> Results in Harpin-dependent Hypersensitive Cell Death in Response to <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

Active defense mechanisms of plants against pathogens often include a rapid plant cell death known as the hypersensitive cell death (HCD). Hypersensitive response-assisting protein (HRAP) isolated from sweet pepper intensifies the harpin_Pss-mediated HCD. Here we demonstrate that constitutive expression of the hrap gene in Arabidopsis results in an enhanced disease resistance towards soft rot pathogen, E. carotovora subsp. carotovora. This resistance was due to the induction of HCD since different HCD markers viz. Athsr3, Athsr4, ion leakage, H₂O₂ and protein kinase were induced. One of the elicitor harpin proteins, HrpN, from Erwinia carotovora subsp. carotovora was able to induce a stronger HCD in hrap-Arabidopsis than non-transgenic controls. To elucidate the role of HrpN, we used E. carotovora subsp. carotovora defective in HrpN production. The hrpN⁻ mutant did not induce disease resistance or HCD markers in hrap-Arabidopsis. These results imply that the disease resistance of hrap-Arabidopsis against a virulent pathogen is harpin dependent.     

Disease resistance gene homologs correlate with disease resistance loci of Arabidopsis thaliana

The disease resistance genes RPS2 of Arabidopsis and N of tobacco, among other recently cloned resistance genes, share several conserved sequences. Degenerate oligonucleotide primers, based on conserved sequences in the nucleotide binding site (NBS) and a weak hydrophobic domain of RPS2 and N, were used to amplify homologous sequences from Arabidopsis thaliana. Amplification products were obtained that were similar in sequence to the disease resistance genes RPS2, RPM1, N and L6. The Arabidopsis CIC-YAC library was used to identify the position of the disease resistance homologs on the Arabidopsis genome. Their map positions could be correlated with the disease resistance loci RPS5, RAC1, RPP9, CAR1, RPP7, RPW2, RPP1, RPP10, RPP14, RPP5, RPP4, RPS2, RPW6, HRT, RPS4, RPP8, RPP21, RPP22, RPP23, RPP24 and TTR1. This method was therefore not only successful in the identification of sequences located within gene clusters that are involved in disease resistance, but could also contribute to the cloning of disease resistance genes from Arabidopsis.     

A geminivirus-induced gene silencing system for gene function validation in cassava

We have constructed an African cassava mosaic virus (ACMV) based gene-silencing vector as a reverse genetics tool for gene function analysis in cassava. The vector carrying a fragment from the Nicotiana tabacumsulfur gene (su), encoding one unit of the chloroplast enzyme magnesium chelatase, was used to induce the silencing of the cassava orthologous gene resulting in yellow–white spots characteristic of the inhibition of su expression. This result suggests that well developed sequence databases from model plants like Arabidopsis thaliana, Nicotiana benthamiana, N. tabacum, Lycopersicon esculentum and others could be used as a major source of information and sequences for functional genomics in cassava. Furthermore, a fragment of the cassava CYP79D2endogenous gene, sharing 89% homology with CYP79D1endogenous gene was inserted into the ACMV vector. The resultant vector was inducing the down regulation of the expression of these two genes which catalyze the first-dedicated step in the synthesis of linamarin, the major cyanogenic glycoside in cassava. At 21 days post-inoculation (dpi), a 76% reduction of linamarin content was observed in silenced leaves. Using transgenic plants expressing antisense RNA of CYP79D1and CYP79D2, Siritunga and Sayre (2003) obtained several lines with a reduction level varying from 60% to 94%. This result provides the first example of direct comparison of the efficiency of a virus-induced gene silencing (VIGS) system and the transgenic approach for suppression of a biosynthetic pathway. The ACMV VIGS system will certainly be a complement and in some cases an alternative to the transgenic approach, for gene discovery and gene function analysis in cassava.     

Resistance in tomato to Xanthomonas campestris pv vesicatoria is determined by alleles of the pepper-specific avirulence gene avrBs3

Bacterial spot disease of tomato and pepper caused by Xanthomonas campestris pv vesicatoria is prevented by resistance genes in the plant that match genes for avirulence in the bacterium. Based on DNA homology to the avirulence gene avrBs3, which induces the resistance response on pepper, we have isolated another avirulence gene from X. c. vesicatoria, designated avrBs3-2. This gene differs in specificity from avrBs3 in inducing the hypersensitive response on tomato but not on pepper. Sequence analysis of the avrBs3-2 gene revealed a high degree of conservation: the 3480 by open reading frame contains an internal region of 17.5 nearly identical 102 bp repeat units that differ in their order from those present in the avrBs3 gene. The coding region is 97% identical to avrBs3 and expresses constitutively a 122 kDa protein, thus representing a natural allele of this gene. The previously isolated 1.7 kb avrBsP gene from X. c. vesicatoria is 100% identical to the corresponding avrBs3-2 sequence, indicating that these genes might be identical. Interestingly, derivatives of avrBs3-2 lacking the C-terminal region and part of the repetitive region are still able to confer incompatibility in tomato. The avrBs3-2 gene is compared with the sequence of avrBs3 derivatives generated by deletion of repeat units that also have avirulence activity on tomato. Both genes, avrBs3 and avrBs3-2, are flanked by a 62 by long inverted repeat, which prompts speculations about the origin of the members of the avrBs3 gene family.     

致病疫霉效应蛋白Pi16275的功能研究

[目的]分析致病疫霉效应蛋白Pi16275的超量表达对病原菌致病性的影响,明确Pi16275的亚细胞定位,筛选Pi16275在植物中的互作靶标蛋白及靶标蛋白在抵御病原菌侵染过程中的作用,初步揭示Pi16275在病原菌侵染植物过程中的作用机制.[方法]利用农杆菌介导的烟草瞬时表达系统在烟草叶片表皮细胞中瞬时表达Pi162...     

木薯UGT41基因对细菌性枯萎病的抗性研究北大核心CSCD

糖基转移酶广泛存在于植物中,其中UDP依赖型糖基转移酶(UDP-glycosyltransferases,UGTs)基因家族是糖基转移酶中的一大类。该研究以华南124木薯品种(Manihot esculenta cv.SC124)为材料,利用RT-PCR技术克隆木薯MeUGT41基因,以病毒诱导干扰木薯MeUGT41基因的表达量,并对基因干扰植株进行细菌性枯萎病抗性评价,为研究MeUGT41基因在木薯抵抗细菌性枯萎病的抗病机理奠定基础。结果表明:(1)地毯草黄单胞菌(Xamthomonas axonopodis pv.Manihotis,Xam)可显著诱导木薯MeUGT41基因表达。(2)成功构建MeUGT41的病毒诱导基因沉默(VIGS)载体,将干扰载体转化至木薯叶片进行MeUGT41基因沉默,荧光定量PCR检测结果显示,木薯叶片中MeUGT41基因的表达量显著下降。(3)Xam侵染实验表明,干扰抑制MeUGT41基因表达可显著降低木薯植株叶片对Xam病菌侵染的抵抗能力。研究认为,木薯叶片中MeUGT41基因具有抵抗Xam病菌侵染的能力。     

The tomato Orion locus comprises a unique class of <Emphasis Type="Italic">Hcr9</Emphasis> genes

Resistance against the tomato fungal pathogen Cladosporium fulvum is often conferred by Hcr9 genes (Homologues of the C. fulvum resistance gene Cf-9) that are located in the Milky Way cluster on the short arm of chromosome 1. These Hcr9 genes mediate recognition of fungal avirulence gene products. In contrast, the resistance gene Cf-Ecp2 mediates recognition of the virulence factor Ecp2 and is located in the Orion (OR) cluster on the short arm of chromosome 1. Here, we report the map- and homology-based cloning of the OR Hcr9 cluster. A method was optimised to generate clone-specific fingerprint data that were subsequently used in the efficient calculation of genomic DNA contigs. Three Hcr9s were identified as candidate Cf-Ecp2 genes. By PCR-based cloning using specific OR sequences, orthologous Hcr9 genes were identified from different Lycopersicon species and haplotypes. The OR Hcr9s are very homologous. However, based on the relative low sequence homology to other Hcr9s, the OR Hcr9s are classified as a new subgroup.Data deposition: The sequence of the Cf-Ecp2 Hcr9 gene cluster and the orthologous Hcr9 sequences have been deposited in the GenBank database (accession No. AY639600..AY639604)     

Calmodulin-like Proteins from <Emphasis Type="Italic">Arabidopsis</Emphasis> and Tomato are Involved in Host Defense Against <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tomato</Emphasis>

Complex signal transduction pathways underlie the myriad plant responses to attack by pathogens. Ca²⁺ is a universal second messenger in eukaryotes that modulates various signal transduction pathways through stimulus-specific changes in its intracellular concentration. Ca²⁺-binding proteins such as calmodulin (CaM) detect Ca²⁺ signals and regulate downstream targets as part of a coordinated cellular response to a given stimulus. Here we report the characterization of a tomato gene (APR134) encoding a CaM-related protein that is induced in disease-resistant leaves in response to attack by Pseudomonas syringae pv. tomato. We show that suppression of APR134 gene expression in tomato (Solanum lycopersicum), using virus-induced gene silencing (VIGS), compromises the plant’s immune response. We isolated APR134-like genes from Arabidopsis, termed CML42 and CML43, to investigate whether they serve a functionally similar role. Gene expression analysis revealed that CML43 is rapidly induced in disease-resistant Arabidopsis leaves following inoculation with Pseudomonas syringae pv. tomato. Overexpression of CML43 in Arabidopsis accelerated the hypersensitive response. Recombinant APR134, CML42, and CML43 proteins all bind Ca²⁺ in vitro. Collectively, our data support a role for CML43, and APR134 as important mediators of Ca²⁺-dependent signals during the plant immune response to bacterial pathogens. This work was supported by a research grant (WAS) and postgraduate scholarships (DC, SLD) from the Natural Science and Engineering Research Council of Canada, the National Science Foundation (IBN-0109633; GBM), and the Swedish Research Council (SKE).     

The Cladosporium fulvum resistance gene Cf-ECP3 is part of the Orion cluster on the short arm of tomato Chromosome 1

The resistance of tomato to the pathogenic fungus Cladosporiumfulvum complies with the gene-for-gene relationship. Race specificresistance is based on Cf-gene mediated recognition ofsecreted avirulence products, resulting in a hypersensitive response (HR).Besides the avirulence gene products, C. fulvum secretes anumber of extra cellular proteins (ECPs) into the apoplast. Two L.esculentum accessions have previously been identified that reactedwith a HR upon injection with purified ECP3. The corresponding resistance genedesignated Cf-ECP3 was mapped by using an F₂population composed of 192 plants from the cross of susceptible MoneyMaker toresistant L. esculentum G1.1153.Cf-ECP3 inherited monogenically, cosegragated with theChromosome 1 Cleaved Amplified Polymorphic Sequence (CAPS) marker CT116 and wasmapped accurately at Orion, a locus harbouring Cf-ECP2 inother genotypes. RFLP anaysis with a Cf-9 probe furtherdemonstrated cosegregation of Cf-ECP3 with anHcr9 (Homologue of Cladosporiumfulvumresistance gene Cf-9) indicating that this gene is likelyan Hcr9. Thus in addition to the Milky Way locusharbouringthe Cf-4, Cf-4A andCf-9 resistance genes targeted against AVR4, AVR4A andAVR9, Orion is another complex locus on the short arm of Chromosome 1 thatharbours at least two functional Cf-genes,Cf-ECP2 and Cf-ECP3, targeted againstthe fungal excreted proteins ECP2 and ECP3.     

A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus

The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by the Madagascar periwinkle (Catharanthus roseus). Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003–0.01% yields. Metabolic engineering efforts to either improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. A VIGS method was developed herein to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro.     

A VIGS screen identifies immunity in the Arabidopsis Pla‐1 accession to viruses in two different genera of the Geminiviridae

Geminiviruses are DNA viruses that cause severe crop losses in different parts of the world, and there is a need for genetic sources of resistance to help combat them. Arabidopsis has been used as a source for virus‐resistant genes that derive from alterations in essential host factors. We used a virus‐induced gene silencing (VIGS) vector derived from the geminivirus Cabbage leaf curl virus (CaLCuV) to assess natural variation in virus–host interactions in 190 Arabidopsis accessions. Silencing of CH‐42, encoding a protein needed to make chlorophyll, was used as a visible marker to discriminate asymptomatic accessions from those showing resistance. There was a wide range in symptom severity and extent of silencing in different accessions, but two correlations could be made. Lines with severe symptoms uniformly lacked extensive VIGS, and lines that showed attenuated symptoms over time (recovery) showed a concomitant increase in the extent of VIGS. One accession, Pla‐1, lacked both symptoms and silencing, and was immune to wild‐type infectious clones corresponding to CaLCuV or Beet curly top virus (BCTV), which are classified in different genera in the Geminiviridae. It also showed resistance to the agronomically important Tomato yellow leaf curl virus (TYLCV). Quantitative trait locus mapping of a Pla‐1 X Col‐0 F₂ population was used to detect a major peak on chromosome 1, which is designated gip‐1 (geminivirus immunity Pla‐1‐1). The recessive nature of resistance to CaLCuV and the lack of obvious candidate genes near the gip‐1 locus suggest that a novel resistance gene(s) confers immunity.     

棉花miR397 LAC4模块调控棉铃虫抗性研究

miRNA(microRNA)通过调控其靶标基因在植物的生长、发育和抗逆过程中扮演着重要的角色。该研究采用分子生物学和生物化学等方法,探讨棉花miR397 LAC4参与植株木质素生物合成和对棉铃虫抗性响应机制。结果发现：(1)棉花miR397(ghr miR397)在转录后调控漆酶基因(GhLAC4)的表达,GhLAC4属于蓝铜氧化酶家族,通过调控木质素合成,抵御棉铃虫入侵棉花。(2)GUS报告基因融合表达和酶活性测定表明,ghr miR397在转录后切割靶标基因GhLAC4抑制其表达。(3)利用VIGS(virus induced gene silencing)技术在棉花中沉默和过表达ghr miR397,棉铃虫抗性检测分析表明,沉默miR397表达会增加棉花对棉铃虫的抗性,但过表达ghr miR397则会降低棉花的抗性。(4)选择性和非选择性棉铃虫实验分析、组织化学染色和木质素含量测定表明,沉默GhLAC4表达会减少木质素的积累,增加棉花对棉铃虫的敏感性。研究表明,ghr miR397 GhLAC4模块共同微调棉花木质素合成来参与棉花抗虫性调控,同时也为棉花抗虫育种提供了新思路。     

Use of Arabidopsis thaliana and Pseudomonas syringae in the Study of Plant Disease Resistance and Tolerance

The interaction between Arabidopsis thaliana and the bacterium Pseudomonas syringae is being developed as a model experimental system for plant pathology research. Race-specific ("gene-for-gene") resistance has been demonstrated for this interaction, and pathogen genes that determine avirulence have been isolated and characterized. Because certain lines of both Arabidopsis and soybean are resistant to bacteria carrying the avirulence genes avrRpt2 and avrB, extremely similar pathogen recognition mechanisms are apparently present in these two plant species. Isogenic bacterial strains that differ by the presence of single avirulence genes are being used to analyze plant resistance. Plant resistance genes have been identified in crosses between resistant and susceptible lines. The extensive map-based cloning tools available in Arabidopsis are being used to isolate these resistance genes. In a related project, ethylene-insensitive Arabidopsis mutants are being used to examine the role of ethylene in disease development. Ethylene apparently mediates symptom formation in susceptible plants and is not required for resistance, suggesting possible strategies for enhancement of disease tolerance in crops.