Avirulent Pathotypes Research Articles

A Pleiotropic Drug Resistance Transporter TaABCG36 Contributes to Defense against Puccinia triticina in Triticum aestivum

ABC transporters play important roles in plant growth and resistance to abiotic and biotic stresses. Here, we showed that the TaABCG36 gene positively regulates leaf rust resistance in the wheat line Thatcher + Lr19 (TcLr19) when challenged with an avirulent pathotype of Puccinia triticina (Pt). The TaABCG36 gene was cloned from genomic DNA and cDNA from wheat line TcLr19. The clone was 6730 bp in gDNA and 4365 bp in cDNA for this gene. It encoded an ABC transporter with 1454 amino acids in length. BLASTp analysis indicated a considerable identity ABC transporter G family member 36 with Aegilops tauschii subsp. strangulata, Triticum dicoccoides, and T. aestivum; thus, we named the gene TaABCG36. TaABCG36 was proved to be a plasma transmembrane protein by bioinformatic analysis and subcellular localization of the TaABCG36–GFP fusion protein. The expression of TaABCG36 in wheat leaves reached a peak at 72 h post-inoculation by Pt avirulence pathotype, and the expression was also induced by phytohormone treatments of salicylic acid (SA), abscisic acid (ABA), and methyl jasmonate (MeJA). Three fragments (V1–V3) of the TaABCG36 gene were introduced to the BSMV-VIGS vector and, thus, silenced the expression of TaABCG36 in the wheat line TcLr19. All the three BSMV:VIGS-infected plants showed reaction type “3” to Pt pathotype THTS, which was fully avirulent on TcLr19 (infection type “0”). Histopathological observation showed that silencing of TaABCG36 facilitated the formation of haustorial mother cells (HMC) and mycelial growth, implying that TaABCG36 plays a positive role in the response of TcLr19 against THTS. These results provide molecular insight into the interaction between Pt and its wheat host and identify a potential target for engineering resistance in wheat to damaging pathogen of Pt.

A Chloroplast-Localized Glucose-6-Phosphate Dehydrogenase Positively Regulates Stripe Rust Resistance in Wheat.

Glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme of the pentose phosphate pathway (PPP), plays a pivotal role in plant stress responses. However, the function and mechanism of G6PDHs in crop plants challenged by fungal pathogens remain poorly understood. In this study, a wheat G6DPH gene responding to infection by Puccinia striiformis f. sp. tritici (Pst), designated TaG6PDH2, was cloned and functionally identified. TaG6PDH2 expression was significantly upregulated in wheat leaves inoculated with Pst or treated with abiotic stress factors. Heterologous mutant complementation and enzymatic properties indicate that TaG6PDH2 encodes a G6PDH protein. The transient expression of TaG6PDH2 in Nicotiana benthamiana leaves and wheat protoplasts revealed that TaG6PDH2 is a chloroplast-targeting protein. Silencing TaG6PDH2 via the barley stripe mosaic virus (BSMV)-induced gene silencing (VIGS) system led to compromised wheat resistance to the Pst avirulent pathotype CYR23, which is implicated in weakened H2O2 accumulation and cell death. In addition, TaG6PDH2 was confirmed to interact with the wheat glutaredoxin TaGrxS4. These results demonstrate that TaG6PDH2 endows wheat with increased resistance to stripe rust by regulating reactive oxygen species (ROS) production.

The barley leaf rust resistance gene Rph3 encodes a predicted membrane protein and is induced upon infection by avirulent pathotypes of Puccinia hordei

Leaf rust, caused by Puccinia hordei, is an economically significant disease of barley, but only a few major resistance genes to P. hordei (Rph) have been cloned. In this study, gene Rph3 was isolated by positional cloning and confirmed by mutational analysis and transgenic complementation. The Rph3 gene, which originated from wild barley and was first introgressed into cultivated Egyptian germplasm, encodes a unique predicted transmembrane resistance protein that differs from all known plant disease resistance proteins at the amino acid sequence level. Genetic profiles of diverse accessions indicated limited genetic diversity in Rph3 in domesticated germplasm, and higher diversity in wild barley from the Eastern Mediterranean region. The Rph3 gene was expressed only in interactions with Rph3-avirulent P. hordei isolates, a phenomenon also observed for transcription activator-like effector-dependent genes known as executors conferring resistance to Xanthomonas spp. Like known transmembrane executors such as Bs3 and Xa7, heterologous expression of Rph3 in N. benthamiana induced a cell death response. The isolation of Rph3 highlights convergent evolutionary processes in diverse plant-pathogen interaction systems, where similar defence mechanisms evolved independently in monocots and dicots.

Transcriptomic Analysis of Resistant and Susceptible Responses in a New Model Root-Knot Nematode Infection System Using Solanum torvum and Meloidogyne arenaria.

Root-knot nematodes (RKNs) are among the most devastating pests in agriculture. Solanum torvum Sw. (Turkey berry) has been used as a rootstock for eggplant (aubergine) cultivation because of its resistance to RKNs, including Meloidogyne incognita and M. arenaria. We previously found that a pathotype of M. arenaria, A2-J, is able to infect and propagate in S. torvum. In vitro infection assays showed that S. torvum induced the accumulation of brown pigments during avirulent pathotype A2-O infection, but not during virulent A2-J infection. This experimental system is advantageous because resistant and susceptible responses can be distinguished within a few days, and because a single plant genome can yield information about both resistant and susceptible responses. Comparative RNA-sequencing analysis of S. torvum inoculated with A2-J and A2-O at early stages of infection was used to parse the specific resistance and susceptible responses. Infection with A2-J did not induce statistically significant changes in gene expression within one day post-inoculation (DPI), but afterward, A2-J specifically induced the expression of chalcone synthase, spermidine synthase, and genes related to cell wall modification and transmembrane transport. Infection with A2-O rapidly induced the expression of genes encoding class III peroxidases, sesquiterpene synthases, and fatty acid desaturases at 1 DPI, followed by genes involved in defense, hormone signaling, and the biosynthesis of lignin at 3 DPI. Both isolates induced the expression of suberin biosynthetic genes, which may be triggered by wounding during nematode infection. Histochemical analysis revealed that A2-O, but not A2-J, induced lignin accumulation at the root tip, suggesting that physical reinforcement of cell walls with lignin is an important defense response against nematodes. The S. torvum-RKN system can provide a molecular basis for understanding plant-nematode interactions.

Comparative Transcriptomics and RNA-Seq-Based Bulked Segregant Analysis Reveals Genomic Basis Underlying Cronartium ribicola vcr2 Virulence.

Breeding programs of five-needle pines have documented both major gene resistance (MGR) and quantitative disease resistance (QDR) to Cronartium ribicola (Cri), a non-native, invasive fungal pathogen causing white pine blister rust (WPBR). WPBR is one of the most deadly forest diseases in North America. However, Cri virulent pathotypes have evolved and can successfully infect and kill trees carrying resistance (R) genes, including vcr2 that overcomes MGR conferred by the western white pine (WWP, Pinus monticola) R gene (Cr2). In the absence of a reference genome, the present study generated a vcr2 reference transcriptome, consisting of about 20,000 transcripts with 1,014 being predicted to encode secreted proteins (SPs). Comparative profiling of transcriptomes and secretomes revealed vcr2 was significantly enriched for several gene ontology (GO) terms relating to oxidation-reduction processes and detoxification, suggesting that multiple molecular mechanisms contribute to pathogenicity of the vcr2 pathotype for its overcoming Cr2. RNA-seq-based bulked segregant analysis (BSR-Seq) revealed genome-wide DNA variations, including about 65,617 single nucleotide polymorphism (SNP) loci in 7,749 polymorphic genes shared by vcr2 and avirulent (Avcr2) pathotypes. An examination of the distribution of minor allele frequency (MAF) uncovered a high level of genomic divergence between vcr2 and Avcr2 pathotypes. By integration of extreme-phenotypic genome-wide association (XP-GWAS) analysis and allele frequency directional difference (AFDD) mapping, we identified a set of vcr2-associated SNPs within functional genes, involved in fungal virulence and other molecular functions. These included six SPs that were top candidate effectors with putative activities of reticuline oxidase, proteins with common in several fungal extracellular membrane (CFEM) domain or ferritin-like domain, polysaccharide lyase, rds1p-like stress responsive protein, and two Cri-specific proteins without annotation. Candidate effectors and vcr2-associated genes provide valuable resources for further deciphering molecular mechanisms of virulence and pathogenicity by functional analysis and the subsequent development of diagnostic tools for monitoring the virulence landscape in the WPBR pathosystems.

Changes in Cytochrome b Gene Expression in Cochliobolus sativus Induced by Triadimefon, a Triazole Fungicide

Introduction: Spot blotch, caused by Cochliobolus sativus, is most effectively managed using fungicide applications, including triadimefon (TDM) a triazole compound. C. sativus posses a great concern as it might develop resistance against fungicides like TDM due to its high genetic variability, short life cycle, and abundant inoculum production. Therefore, to better understand the mechanisms of TDM resistance initiated by C. sativus, changes in cytochrome b (cytb) gene in virulent and avirulent pathotypes were evaluated at early time points of TDM treatments. Materials and Methods: C. sativus sensitivity to TDM was determined by measuring the radial growth of each pathotype on PDA plates. Additionally, RNA was isolated from mycelia of each pathotype at 24, 48, 72 and 96 hours post fungicide treatments and used for cDNA synthesis. Cytb was verified using quantitative reverse transcriptase PCR (qRT-PCR). Results: Data showed that the maximum mycelial growth inhibition by 50% (EC50) for both pathotypes was recorded 48h at 0.25 µg/ml TDM treatment. The qRT-PCR revealed that cytb expression increased in both virulent and avirulent pathotypes at 24h post TDM treatments in comparison with non-treated controls. The most outstanding differences in cytb expression were7.69 and 2.88-fold in the virulent and avirulent pathotypes, respectively, 48h of 0.25 µg/ml TDM treatment. Conclusions: According to findings, it is possible to propose that cytb gene might play a role in signaling events during C. sativus exposure to commercial triazole fungicide.

Double dose efficiency of the yellow rust resistance gene Yr17 in bread wheat lines

AbstractYellow rust, caused by Puccinia striiformis f. sp. tritici, is one of the most severe wheat disease worldwide. Crop losses have ranged from 10% to 70% and up to 100% in extreme conditions. Eighty‐two resistance genes, designated Yr, have been identified. Among them, Yr17 derived from Aegilops ventricosa and located on chromosome 2A has been widely used in wheat breeding. However, it had been overcome already. Through recombination of the Ae. ventricosa Yr17‐carrying 6Nv chromosome with 2D of wheat, we introduced Yr17 onto chromosome 2D. Then, lines carrying Yr17 on both 2A and 2D were generated. Seedlings of the latter, as well as those carrying a single dose of Yr17 either on 2A or on 2D, were inoculated with virulent or avirulent strains on wheat seedlings. The different genotypes were fully susceptible for the two pathotypes that are virulent on Yr17. In the case of avirulent pathotypes, the Yr17 double dose lines were fully resistant, while those with the Yr17 gene only on either 2A or 2D had intermediate resistance reactions towards one or the other or both pathotypes.

Molecular mapping of leaf rust resistance gene LrL224 in Chinese wheat cultivar L224-3

Leaf rust, caused by Puccinia triticina, is a major wheat disease worldwide. The chinese wheat cultivar L224-3 showed high resistance to most of P. triticina pathotypes in the seedling and adult stage. Identifying and mapping the leaf rust resistance gene(s) in L224-3 is very useful for breeding leaf rust resistant wheat cultivars. In the present study, the wheat cultivar L224-3 and thirty-six lines with known leaf rust resistance genes were inoculated with 15 pathotypes at the seedling stage for gene postulation. A total of 144 F<sub>2:3</sub> lines from the cross L224-3 × Zhengzhou 5389 were inoculated with the pathotype FHBQ for leaf rust genetic analysis at the seedling stage. A total of 1276 SSR (simple sequence repeat) markers and the STS (sequence tagged-site) marker ω-secali/Glu-B3 were used to test the parents, resistant and susceptible bulks. The polymorphic markers were used to genotype the F<sub>2:3</sub> populations. L224-3 was highly resistant to all Lr26 avirulent pathotypes, showing the presence of Lr26 in L224-3. The presence of Lr26 in L224-3 was also confirmed by the molecular marker ω-secalin/Glu-B3. Due to resistance to some Lr26 virulent pathotypes, L224-3 may carry another resistance gene. Based on the genetic analysis using the pathotype FHBQ with virulence to Lr26 the resistance of L224-3 was controlled by a single dominant gene, tentatively designated LrL224. Four SSR markers (barc8, gwm582, wmc419, and wmc694) and one STS marker (ω-secali/Glu-B3) on 1B were closely linked to LrL224. The two flanking SSR loci were barc8 and gwm582, with the genetic distances of 4.3 and 4.6 cM, respectively. LrL224 was located on 1BL, and it showed different seedling reactions with other genes on 1B. Therefore LrL224 is likely to be a new leaf rust resistance gene.

Saturated genic SNP mapping identified functional candidates and selection tools for the Pinus monticola Cr2 locus controlling resistance to white pine blister rust.

SummaryMolecular breeding incorporates efficient tools to increase rust resistance in five‐needle pines. Susceptibility of native five‐needle pines to white pine blister rust (WPBR), caused by the non‐native invasive fungus Cronartium ribicola (J.C. Fisch.), has significantly reduced wild populations of these conifers in North America. Major resistance (R) genes against specific avirulent pathotypes have been found in several five‐needle pine species. In this study, we screened genic SNP markers by comparative transcriptome and genetic association analyses and constructed saturated linkage maps for the western white pine (Pinus monticola) R locus (Cr2). Phenotypic segregation was measured by a hypersensitive reaction (HR)‐like response on the needles and disease symptoms of cankered stems post inoculation by the C. ribicola avcr2 race. SNP genotypes were determined by HRM‐ and TaqMan‐based SNP genotyping. Saturated maps of the Cr2‐linkage group (LG) were constructed in three seed families using a total of 34 SNP markers within 21 unique genes. Cr2 was consistently flanked by contig_2142 (encoding a ruvb‐like protein) and contig_3772 (encoding a delta‐fatty acid desaturase) across the three seed families. Cr2 was anchored to the Pinus consensus LG‐1, which differs from LGs where other R loci of Pinus species were mapped. GO annotation identified a set of NBS‐LRR and other resistance‐related genes as R candidates in the Cr2 region. Association of one nonsynonymous SNP locus of an NBS‐LRR gene with Cr2‐mediated phenotypes provides a valuable tool for marker‐assisted selection (MAS), which will shorten the breeding cycle of resistance screening and aid in the restoration of WPBR‐disturbed forest ecosystems.

TaRar1 Is Involved in Wheat Defense against Stripe Rust Pathogen Mediated by YrSu.

RAR1 is a eukaryotic zinc-binding protein first identified as required for race-specific resistance to powdery mildew in barley. To study the function of TaRAR1 involvement in wheat (Triticum aestivum L.) defense against the infection of stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst), we identified and cloned three wheat homeologous genes highly similar to the barley HvRar1, designated as TaRar1-2A, TaRar1-2B, and TaRar1-2D. The three TaRAR1 proteins all contain two conserved cysteine-and histidine-rich domains (CHORD-I and -II) shared by known RAR1-like proteins. Characterization of TaRar1 expression revealed that the expression was tissue-specific and up-regulated in wheat during stripe rust infection. Moreover, the transcription of TaRar1 was induced by methyl jasmonate, ethylene, and abscisic acid hormones. The same results were observed with drought and wound treatments. After TaRar1 was silenced in wheat cultivar Suwon11 containing the stripe rust resistance gene YrSu, the endogenous salicylic acid (SA) level, the hydrogen peroxide (H2O2) accumulation and the degree of hypersensitive response (HR) were significantly decreased, and the resistance to the avirulent pathotype of stripe rust was compromised. Meanwhile, the expression of catalase, an enzyme required for H2O2-scavenging, was up-regulated. Taken together, we concluded that TaRar1 is involved in wheat defense against stripe rust mediated by YrSu, and the defense was through SA to influence reactive oxygen species accumulation and HR.

English

Barley nurseries comprising 820 lines with 479 unique pedigrees sourced from the International Centre for Agricultural Research in the Dry Areas (ICARDA) were screened for seedling and adult plant resistance (APR) against Australian isolates of barley leaf rust pathogen Puccinia hordei Otth. Ninety three percent of the lines were postulated to carry the seedling leaf rust resistance gene Rph3 based on their susceptibility in the greenhouse and field against Rph3 virulent pathotype and resistance to Rph3 avirulent pathotypes. The remaining lines showed either presence of uncharacterised seedling resistance (1%) and uncharacterised APR (1%). Five percent of lines were susceptible at both seedling and adult plant growth stages. Of the six lines identified to carry uncharacterised APR, three likely carried Rph20 based on the presence of the Rph20-linked marker bPb-0837. The results suggested that most of the ICARDA germplasm tested is not suitable for leaf rust resistance in Australia due to the presence of virulence for Rph3. Lines carrying uncharacterised seedling resistance and APR are potentially new sources of resistance, and are recommended for genetic analysis. Key words: Hordeum vulgare, Puccinia hordei, adult plant resistance, Rph3.

Virulence differences among Sclerotinia sclerotiorum isolates determines host cotyledon resistance responses in Brassicaceae genotypes

Differences in Sclerotinia rot (SR) disease severity, caused by two categorized pathotypes and one more recent isolate of S. sclerotiorum and measured in terms of cotyledon lesion diameter, were studied across diverse Brassicaceae hosts to characterize host response and pathogen virulence. There were significant differences (P ≤0.001) between genotypes, isolates and a significant genotype x isolate interaction. The mean diameter of cotyledon lesions ranged from 5 mm in the most resistant genotypes (e.g., Brassica juncea Ringot I and Seeta) to ≥ 13.6 mm in the most susceptible genotypes (e.g., B. tournefortii Wild turnip #1 and #2, Sisymbrium irio London rocket Wild #1 and #2, and B. nigra 4381). Responses, in at least one experiment for some B. juncea (e.g., Seeta, Ringot I) and Raphanus sativus (e.g., Colonel) genotypes, were generally highly resistant irrespective of the isolate used, making them ideal sources of resistance to exploit for developing new varieties with more effective resistance to SR across multiple pathotypes of this pathogen. In contrast, some other genotypes showed significant isolate dependency, with high levels of resistance against one isolate (e.g., B. napus Charlton against the WW4 isolate; B. napus Oscar against the ‘Cabbage’ isolate) but quite susceptible to other isolates (e.g., B. napus Charlton against the ‘Cabbage’ and MBRS1 isolates; B. napus Oscar against the WW4 isolate). These findings highlight the value from using pathotypes of different physiological specialization in screening programs to identify host resistance that is durable across multiple pathotypes. Distinct host resistance symptom types were reported for the first time on some genotypes against isolate WW4; including a distinct yellow halo observed around lesions on B. napus RQ001, indicative of leaf senescence involved in programmed cell death (PCD); a distinct dark brown margin observed around lesions on R. sativus, indicative of a hypersensitive response (HR); and the HR ‘flecking’ on Sinapis alba Concerta and B. juncea Seeta. That WW4 was the most pathogenic isolate for genotypes such as B. juncea Hetianyoucai and B. napus Oscar that showed high level resistance to the ‘Cabbage’ isolate and intermediate resistance to MBRS-1, dispels previously held views that WW4 was a largely avirulent pathotype of little consequence. Rather, isolate WW4 offers unique opportunities to investigate HR and PCD host resistance responses to S. sclerotiorum in Brassicaceae.

Changes in virulence frequencies and higher fitness of simple pathotypes in the Czech population of Blumeria graminis f.sp. hordei

Dreiseitl A. (2015): Changes in virulence frequencies and higher fitness of simple pathotypes in the Czech population of Blumeria graminis f.sp. hordei. Plant Protect. Sci., 51: 67–73. The employment of specific genes, which determine host resistance to avirulent pathotypes of the pathogen, is not recommended for protecting barley against powdery mildew ( Blumeria graminis f.sp. hordei). However, most barley varieties still possess specific resistance genes to this pathogen. The effectiveness of such resistances was determined by assessing virulence frequency using randomly collected Czech population samples of 521 pathogen isolates from the air tested on 29 barley differential varieties over a four-year period. Most virulence frequencies did not show major changes, but the frequencies on the winter cv. Laverda and the spring cv. Kangoo greatly increased. The highest viru lence frequency in all four years was on the resistance Ru2 in Kompolti 4. The period of sampling the air population and associated changes in the proportion of spores that developed on spring and winter varieties with different resist ances are one of causes of the changes in virulence frequencies. It was also found out that the virulence complexity of pathotypes with the frequency > 1 was markedly lower than the complexity of pathotypes with the frequency of 1. This could indicate the higher fitness of pathotypes with fewer virulences.

The Role of Primary and Secondary Infection in Host Response to Plasmodiophora brassicae

The disease cycle of Plasmodiophora brassicae consists of a primary phase in root hairs followed by a secondary phase in the root cortex and adjacent tissues. However, the role of root hair infection in subsequent cortical infection and development of P. brassicae is not well understood. To examine the role of the primary and secondary stages separately, inoculation studies with resting spores (source of primary zoospores) and secondary zoospores of a virulent and avirulent pathotype were conducted on canola (Brassica napus). The size of secondary zoospores and number of nuclei were also examined. The zoospores were larger (≈9.6 to 14.4 μm) than in previous reports and all were uninucleate. Inoculation with secondary zoospores alone produced both primary and secondary infection, even with the avirulent pathotype. No symptoms developed from inoculation with avirulent primary zoospores but tiny, bead-shaped clubs developed from inoculation with avirulent secondary zoospores. Inoculation with virulent secondary zoospores alone resulted in lower disease severity than inoculation with virulent resting spores alone. The results indicate that recognition of infection by the host and initiation of a response (induction or suppression of resistance) occurs during primary infection, although recognition can also occur during cortical infection and development.

Failure of RPG1 protein to degrade in high-copy Rpg1 transgenic barley lines results in susceptibility to stem rust

Rpg1 is a valuable resistance gene that has protected barley from serious yield losses due to stem rust (Puccinia graminis f. sp. tritici [Pgt]) for over 60 years in the Upper Midwest region of the United States. In previous research, this gene was cloned from cultivar (cv.) Morex and its function validated by transforming the susceptible barley cv. Golden Promise into a resistant cultivar. A single copy of Rpg1 was sufficient to confer stem rust resistance, but we identified high-copy transgenic lines (with four and five copies of Rpg1) that were susceptible at both the seedling and adult plant stages. To elucidate the basis of these susceptible reactions, we selected six transgenic lines with variable copy numbers of Rpg1 and tested them for level of RPG1 protein accumulation, RPG1 protein degradation upon inoculation with the avirulent pathotype Pgt-MCCF, RPG1 phosphorylation in response to the same avirulent pathotype, and elicitation of a hypersensitive response upon infiltration with Pgt effectors RGD-binding protein and VPS9 protein. The stem rust resistant transgenic lines with one and two copies of Rpg1 (G04-271, G04-273 G04-266, and G03-210) reacted very similar to the positive control of cv. Morex with respect to all of the assays. In contrast, the behavior of the stem rust susceptible high-copy lines (G04-287 with four copies and G04-288 with five copies) was different in several respects. First, the level of RPG1 protein was about 1.5–3.0× higher than in the resistant lines and cv. Morex. Second, the protein failed to degrade, unlike the case with the resistant lines and cv. Morex where RPG1 was completely degraded by 28 hpi. Third, the high-copy lines failed to elicit a hypersensitive response after infiltration with the Pgt effectors, whereas the resistant lines did. RPG1 protein from the high-copy transgenic lines appeared to behave normally with respect to its ability to phosphorylate in vivo. However, these two lines over produced apparently functional RPG1 protein, but failed to degrade it normally. Stem rust susceptibility and failure to respond to avirulence factors by the high-copy Rpg1 transgenic lines G04-287 and G04-288 are probably due to the failure to degrade the RPG1 protein.

Wheat BAX inhibitor-1 contributes to wheat resistance to Puccinia striiformis

BAX inihibitor-1 (BI-1) is proposed to be a cell death suppressor conserved in both animals and plants. The ability of BI-1 genes to inhibit programmed cell death (PCD) has been well studied in animals, but the physiological importance of BI-1 in plant-microbe interactions remains unclear. This study characterized BI-1 from wheat infected by Puccinia striiformis f. sp. tritici (Pst). The deduced TaBI-1 protein contained a Bax inhibitor domain and seven transmembrane regions conserved among members of the BI-1 family. Transcription of TaBI-1 was detected in all wheat tissues tested (culms, roots, leaves, anthers, and spikelets). Furthermore, TaBI-1 exhibited positive transcriptional responses to Pst infection and abiotic stresses. Overexpression of TaBI-1 in tobacco blocked Bax-induced cell death. Silencing TaBI-1 in plants of a resistant wheat genotype converted a resistant reaction to a relatively susceptible reaction when inoculated with an avirulent pathotype of the pathogen, and increased the area per infection site, but the percentage of necrotic cells did not change significantly, indicating that TaBI-1, a negative cell death regulator, contributes to wheat resistance to stripe rust. These results provide a better understanding of the molecular mechanism of wheat resistance to stripe rust.

Modulation of reactive oxygen species activities and H2O2 accumulation during compatible and incompatible tomato–root‐knot nematode interactions

Here, the interaction of Melodoigyne incognita virulent and avirulent pathotypes with susceptible and Mi-resistant tomato (Solanum lycopersicon) has been studied. Significant differences in nematode penetration occurred 2 days postinoculation (dpi) and became stable from 3 dpi onwards. The hypersensitive cell response (HR) in resistant plants prevented the installation of the avirulent pathotype. The virulent pathotype overcame the Mi (nematode) resistance and induced feeding sites in root cells without triggering HR. Reactive oxygen species (ROS), visualized by subcellular reduction of nitroblue tetrazolium, accumulated in nematode penetrated cells. Quantitative analyses with dichlorofluorescein indicated that the oxidative burst occurred very early with both pathotypes, with an enhanced rate in hyper-responsive cells. Hydrogen peroxide (H(2)O(2)), detected by cerium chloride reaction, accumulated in the cell walls and especially in cells neighbouring HR. The apoplastic location of cerium perhydroxide indicated that either the plasma membrane or the cell wall was the primary site of the superoxide/H(2)O(2) generator. The data provide evidence, for the first time, for ROS-generated signals and their spatiotemporal expression in the host and nonhost interaction of tomato with nematodes.

Genetic Variation and Virulence on Lr26 in Puccinia triticina

ABSTRACT The genetic relationships between isolates of Puccinia triticina virulent on wheat with the Lr26 resistance gene were studied. The diversity within and between isolates of P. triticina from Israel, Europe, and the United States was determined by virulence on near-isogenic Thatcher lines and by random amplified polymorphic DNA. According to the molecular markers, isolates that were virulent on Lr26 had diversity levels similar to those of Lr26 nonpathogenic isolates. Distances between subpopulations of isolates virulent and avirulent on Lr26 varied and were unrelated to the Lr26 virulence phenotype. Cluster analysis suggested four groups, three of which were closely associated with the geographical origin of the isolates-Israel, the United States, and Europe. All four groups included both Lr26 virulent and avirulent pathotypes. The results showed that Lr26 virulent rust pathotypes are as genetically dissimilar as the rest of the population. The cluster analysis showed that the rust population in Israel includes at least two different subpopulations, both of which contain Lr26 virulent and Lr26 avirulent isolates.

Isolation of pepper mRNAs differentially expressed during the hypersensitive response to tobacco mosaic virus and characterization of a proteinase inhibitor gene

Pepper ( Capsicum annuum L. cv. Bugang) displays a hypersensitive response (HR) upon infection by tobacco mosaic virus (TMV) pathotype P 0. To elucidate molecular mechanisms underlying this response, a cDNA library was constructed and differentially screened between TMV-P 0-inoculated and uninoculated leaves. Of 3260 clones analyzed by reverse RNA slot-blot hybridization, 22 showed up-regulated expression upon TMV-P 0 challenge. Among these, 16 clones showed significantly higher levels of expression in response to the avirulent pathotype TMV-P 0 than to the virulent pathotype TMV-P 1.2. Nucleotide sequence analysis and database searches revealed that the polypeptides deduced from the selected clones displayed significant sequence homology to various known proteins, indicating conservation of the wide range of up-regulated proteins during HR against viral challenge in higher plants. A cDNA clone encoding a putative proteinase inhibitor II ( CaPinII) was selected and further analyzed. Upon TMV-P 0 inoculation, the CaPinII transcript accumulated maximally at 72 h post-inoculation and declined afterwards. In contrast, inoculation with TMV-P 1.2 did not induce CaPinII expression to any detectable level. The CaPinII gene was also expressed in response to the treatment of various chemicals, as well as wounding. Southern blot analysis showed that a small family of genes homologous to the CaPinII gene is present in the pepper genome.

Susceptibility in oats to stem rust induced by co‐infection with leaf rust

Induction of susceptibility in oats to a normally avirulent pathotype of Puccinia graminis f.sp. avenae was studied in the presence of different pathotypes of P. coronata f.sp. avenae. Induction occurred on seedlings only in the presence of a virulent culture of P. coronata avenae and was not dependent on time or order of inoculation of either pathogen. This phenomenon was restricted to seedlings of lines possessing the Pg‐a source of oat stem rust resistance. The specificity of induced susceptibility can be used as a valuable bioassay for screening and identifying Pg‐a. Induced susceptibility occurred only at the seedling stage, and apparently provides no obstacle to the use of Pg‐a as a source of stem rust resistance in oats.