Race Non-specific Resistance Research Articles

A multiple resistance locus on chromosome arm 3BS in wheat confers resistance to stem rust (Sr2), leaf rust (Lr27) and powdery mildew

Sr2 is the only known durable, race non-specific adult plant stem rust resistance gene in wheat. The Sr2 gene was shown to be tightly linked to the leaf rust resistance gene Lr27 and to powdery mildew resistance. An analysis of recombinants and mutants suggests that a single gene on chromosome arm 3BS may be responsible for resistance to these three fungal pathogens. The resistance functions of the Sr2 locus are compared and contrasted with those of the adult plant resistance gene Lr34.

Differential disease resistance response in the barley necrotic mutant nec1

BackgroundAlthough ion fluxes are considered to be an integral part of signal transduction during responses to pathogens, only a few ion channels are known to participate in the plant response to infection. CNGC4 is a disease resistance-related cyclic nucleotide-gated ion channel. Arabidopsis thaliana CNGC4 mutants hlm1 and dnd2 display an impaired hypersensitive response (HR), retarded growth, a constitutively active salicylic acid (SA)-mediated pathogenesis-related response and elevated resistance against bacterial pathogens. Barley CNGC4 shares 67% aa identity with AtCNGC4. The barley mutant nec1 comprising of a frame-shift mutation of CNGC4 displays a necrotic phenotype and constitutively over-expresses PR-1, yet it is not known what effect the nec1 mutation has on barley resistance against different types of pathogens.Resultsnec1 mutant accumulated high amount of SA and hydrogen peroxide compared to parental cv. Parkland. Experiments investigating nec1 disease resistance demonstrated positive effect of nec1 mutation on non-host resistance against Pseudomonas syringae pv. tomato (Pst) at high inoculum density, whereas at normal Pst inoculum concentration nec1 resistance did not differ from wt. In contrast to augmented P. syringae resistance, penetration resistance against biotrophic fungus Blumeria graminis f. sp. hordei (Bgh), the causal agent of powdery mildew, was not altered in nec1. The nec1 mutant significantly over-expressed race non-specific Bgh resistance-related genes BI-1 and MLO. Induction of BI-1 and MLO suggested putative involvement of nec1 in race non-specific Bgh resistance, therefore the effect of nec1on mlo-5-mediated Bgh resistance was assessed. The nec1/mlo-5 double mutant was as resistant to Bgh as Nec1/mlo-5 plants, suggesting that nec1 did not impair mlo-5 race non-specific Bgh resistance.ConclusionsTogether, the results suggest that nec1 mutation alters activation of systemic acquired resistance-related physiological markers and non-host resistance in barley, while not changing rapid localized response during compatible interaction with host pathogen. Increased resistance of nec1 against non-host pathogen Pst suggests that nec1 mutation may affect certain aspects of barley disease resistance, while it remains to be determined, if the effect on disease resistance is a direct response to changes in SA signaling.

Identification of Sources of Resistance to Xanthomonas campestris pv. campestris in Brassica napus Crops.

Black rot, caused by Xanthomonas campestris pv. campestris, is one of the most important diseases affecting Brassica crops worldwide. Nine races have been differentiated in X. campestris pv. campestris, with races 1 and 4 being the most virulent and widespread. The objective of this work was to identify sources of resistance to races 1 and 4 of X. campestris pv. campestris in different Brassica napus crops, mainly in the underexplored pabularia group. Seventy-six accessions belonging to four B. napus groups were screened for resistance to two X. campestris pv. campestris races (1 and 4). The strain of race 1 used in this study was more virulent on the tested materials than the strain of race 4. No race-specific resistance was found to race 1. Most cultivars were susceptible except Russian kale, from the pabularia group, which showed some resistant plants and some other accessions with some partially resistant plants. High levels of race-specific resistance to race 4 were found in the pabularia group, and great variability within accessions was identified. Three improved cultivars (Ragged Jack kale, Friese Gele, and Valle del Oro) and four landraces (Russian kale, MBG-BRS0037, MBG-BRS0041, and MBG-BRS0131) showed plants with some degree of resistance to both races, which may indicate that race-nonspecific resistance is involved. These accessions could be directly used in breeding programs, either as improved cultivars or as donors of race-specific resistance to other Brassica cultivars.

Molecular genetics of race non-specific rust resistance in wheat

Over 150 resistance genes that confer resistance to either leaf rust, stripe rust or stem rust have been catalogued in wheat or introgressed into wheat from related species. A few of these genes from the ‘slow-rusting’ adult plant resistance (APR) class confer partial resistance in a race non-specific manner to one or multiple rust diseases. The recent cloning of two of these genes, Lr34/Yr18, a dual APR for leaf rust and stripe rust, and Yr36, a stripe rust APR gene, showed that they differ from other classes of plant resistance genes. Currently, seven Lr34/Yr18 haplotypes have been identified from sequencing the encoding ATP Binding Cassette transporter gene from diverse wheat germplasm of which one haplotype is commonly associated with the resistance phenotype. The paucity of well characterised APR genes, particularly for stem rust, calls for a focused effort in developing critical genetic stocks to delineate quantitative trait loci, construct specific BAC libraries for targeted APR genes to facilitate robust marker development for breeding applications, and the eventual cloning of the encoding genes.

Race non-specific resistance to rust diseases in CIMMYT spring wheats

Rust diseases continue to cause significant losses to wheat production worldwide. Although the life of effective race-specific resistance genes can be prolonged by using gene combinations, an alternative approach is to deploy varieties that posses adult plant resistance (APR) based on combinations of minor, slow rusting genes. When present alone, APR genes do not confer adequate resistance especially under high disease pressure; however, combinations of 4–5 such genes usually result in “near-immunity” or a high level of resistance. Although high diversity for APR occurs for all three rusts in improved germplasm, relatively few genes are characterized in detail. Breeding for APR to leaf rust and stripe rust in CIMMYT spring wheats was initiated in the early 1970s by crossing slow rusting parents that lacked effective race-specific resistance genes to prevalent pathogen populations and selecting plants in segregating populations under high disease pressure in field nurseries. Consequently most of the wheat germplasm distributed worldwide now possesses near-immunity or adequate levels of resistance. Some semidwarf wheats such as Kingbird, Pavon 76, Kiritati and Parula show high levels of APR to stem rust race Ug99 and its derivatives based on the Sr2-complex, or a combination of Sr2 with other uncharacterized slow rusting genes. These parents are being utilized in our crossing program and a Mexico-Kenya shuttle breeding scheme is used for selecting resistance to Ug99. High frequencies of lines with near-immunity to moderate levels of resistance are now emerging from these activities. After further yield trials and quality assessments these lines will be distributed internationally through the CIMMYT nursery system.

Cellular and transcriptional responses of wheat during compatible and incompatible race‐specific interactions with Puccinia striiformis f. sp. tritici

The initial stages of Puccinia striiformis f. sp. tritici (the causal agent of yellow rust in wheat) infection triggered a hypersensitive cell death (HCD) response in both compatible and Yr1-mediated incompatible interactions, although the response was earlier and more extensive in the incompatible interaction. Later stages of fungal development were only associated with an HCD response in the incompatible interaction, the HCD response being effectively suppressed in the compatible interaction. Cell autofluorescence was seen in mesophyll cells in direct contact with fungal infection hyphae (primary HCD) and in adjacent mesophyll cells (secondary HCD), indicating the activation of cell-to-cell signalling. Microarray analysis identified a number of defence-related transcripts implicated in Yr1-mediated resistance, including classical pathogenesis-related (PR) transcripts and genes involved in plant cell defence responses, such as the oxidative burst and cell wall fortification. A quantitative reverse transcriptase-polymerase chain reaction time course analysis identified a number of defence-related genes, including PR2, PR4, PR9, PR10 and WIR1 transcripts, associated with the latter stages of Yr1-mediated resistance. A meta-analysis comparison of the Yr1-regulated transcriptome with the resistance transcriptomes of the race-specific resistance gene Yr5 and the race-nonspecific adult plant resistance gene Yr39 indicated limited transcript commonality. Common transcripts were largely confined to classic PR and defence-related genes.

Inheritance of red rot resistance in sugarcane (Saccharum sp. hybrids)

Inheritance of resistance to red rot caused by Colletotrichum falcatum Went was studied in progenies from 39 crosses involving 45 parental clones of interspecific and intervarietal origin. The progenies were subjected for screening against red rot under controlled condition testing (CCT) method. In general, crosses involving resistant parents tended to have more number of resistant progenies than those with susceptible parents. Out of 39 crosses investigated for race specific resistance as a qualitative trait, 18 crosses showed simple Mendelian monogenic segregation (vertical resistance). Parent progeny regression analysis suggested that about 50% of the variation in the population could be attributed to horizontal resistance (additive genetic variance). Two crosses involving susceptible parents viz., 971235 (S) × Co 1148 (S) and Co 88028 (S) × Co 775 (S) contributed 28–30% resistant progenies. These transgressive segregants could be used as donor parents in red rot resistance breeding programmes for imparting race non-specific resistance.

Annual Meeting, Winnipeg, Manitoba, 2009

Annual Meeting, Winnipeg, Manitoba, 2009

A Recessive Mutation in Rice Conferring Non-Race-Specific Resistance to Bacterial Blight and Blast

Abstract To understand the basis of broad-spectrum disease resistance in rice, we isolated a gamma-ray-induced IR64 mutant G978 that showed enhanced resistance to blast and bacterial blight. The resistance is quantitative and non-race specific against the bacterial and fungal pathogens. The mutation is inherited as a single recessive gene, designated as Bsdr1 and causes shorter stature relative to the wild type; however, it does not show lesion mimics phenotype under the conditions tested. The mutation was mapped as a quantitative trait locus to a 3.8-Mb region on chromosome 12. By comparing the gene expression profiles of the mutant and wild type, we identified a candidate gene encoding a U-box domain-containing protein. The disrupted gene showed a loss of expression in the mutant and co-segregated with mutant phenotype. The mutant provides a useful tool for investigating the important genes responsible for non-race specific resistance to two distinct diseases.

Diversity of defence mechanisms in plant–oomycete interactions: a case study of Lactuca spp. and Bremia lactucae

Plant pathogenic oomycetes, including biotrophic downy mildews and hemibiotrophs/necrotrophs such as Phytophthora and Pythium, cause enormous economic losses on cultivated crops. Lettuce breeders and growers face the threat of Bremia lactucae, the causal agent of lettuce downy mildew. This pathogen damages leaf tissues and lettuce heads and is also frequent on wild Asteraceae plants. The interactions of Lactuca spp. with B. lactucae (abbr. lettuce–Bremia) display extreme variability, due to a long co-evolutionary history. For this reason, during the last 30 years, the lettuce–Bremia pathosystem has been used as a model for many studies at the population, individual, organ, tissue, cellular, physiological and molecular levels, as well as on genetic variability and the genetics of host–parasite interactions. The first part of this review summarizes recent data on host–parasite specificity, host variability, resistance mechanisms and genetics of lettuce–Bremia interactions. The second part focuses on the development infection structures. Phenotypic expression of infection, behaviour of B. lactucae on leaf surfaces, the process of penetration, development of primary infection structures, hyphae and haustoria are discussed in relation to different resistance mechanisms. In the third part, the components of host resistance and the variability of defence responses are analysed. The role of reactive oxygen species (ROS), antioxidant enzymes, nitric oxide (NO), phenolic compounds, reorganization of cytoskeleton, electrolyte leakage, membrane damage, cell wall disruption, hypersensitive reaction and plant energetics are discussed in relation to defence responses. In general, the extreme variability of interactions between lettuce and Bremia, and their phenotypic expression, results from diversity of the genetic background. Different mechanisms of resistance are conditioned by an orchestra of defence responses at the tissue, cell, and molecular levels. The various events responsible for defence involve a complex interaction of the processes and reactions mentioned above. This review also provides an overview on the timing of pathogen development, host pathological anatomy, cytology and physiology of lettuce–Bremia associations. The significance of these factors on the expression of different resistance mechanisms (non-host and host resistance, race-specific and race non-specific resistance, field resistance) is discussed.

Transcriptome analysis of high-temperature adult-plant resistance conditioned by Yr39 during the wheat-Puccinia striiformis f. sp. tritici interaction.

Stripe rust [caused by Puccinia striiformis Westend. f. sp. tritici Eriks. (Pst)] is a destructive disease of wheat (Triticum aestivum L.) worldwide. High-temperature adult-plant (HTAP) resistance to stripe rust is race non-specific, inherited quantitatively and durable. Previously, we identified and mapped the single Yr39 HTAP stripe rust resistance gene in the spring wheat cultivar Alpowa, which was identified on chromosome 7BL and accounted for 64.2% of the variation in resistance. To identify transcripts associated with Yr39-mediated resistance, we selected two F(7 )recombinant inbred lines (RILs) from an 'Avocet S/Alpowa' cross that differed at the Yr39 locus to represent an incompatible (Yr39) and compatible (yr39) interaction with Pst. Using the Affymetrix Wheat GeneChip, we profiled the transcript changes occurring in flag leaves of these two RILs over a time-course after treatment with Pst urediniospores and mock-inoculation. This time-course study identified 99 induced transcripts that were classified as HTAP resistance-specific. The temporal pattern of transcript accumulation showed a peak at 48 h after infection, which was supported by microscopic observation of fungal development and quantitative PCR assays that showed a rapid increase in fungal biomass after this time in the compatible interaction. More than half (50.5%) of the annotated transcripts specifically induced during HTAP resistance were involved in defence and/or signal transduction, including R gene homologues and transcripts associated with pathogenesis-related protein production, phenylpropanoid biosynthesis and protein kinase signalling. This study represents the first transcript profiling of HTAP resistance to stripe rust in wheat, and we compare our results with other transcript studies of race-specific and race non-specific resistance.

Genetics, Genomics and Breeding of Late Blight and Early Blight Resistance in Tomato

Late blight (LB), caused by the oomycete Phytophthora infestans, and early blight (EB), caused by the fungi Alternaria solani and A. tomatophila, are two common and destructive foliar diseases of the cultivated tomato (Solanum lycopersicum) and potato (Solanum tuberosum) in the United States and elsewhere in the world. While LB can infect and devastate tomato plants at any developmental stages, EB infection is usually associated with plant physiological maturity and fruit load where older senescing plants exhibit greater susceptibility and a heavy fruit set enhances the disease. At present, cultural practices and heavy use of fungicides are the most common measures for controlling LB and EB. Genetic resources for resistance have been identified for both diseases, largely within the tomato wild species, in particular the red-fruited species S. pimpinellifolium and the green-fruited species S. habrochaites. A few race-specific major resistance genes (e.g., Ph-1, Ph-2 and Ph-3) and several race-nonspecific resistance QTLs have been reported for LB. Ph-3 is a strong resistance gene and has been incorporated into many breeding lines of fresh market and processing tomato. However, new P. infestans isolates have been identified which overcome Ph-3 resistance. Recently, a new resistance gene (Ph-5) has been identified, which confers resistance to several pathogen isolates including those overcoming the previous resistance genes. Advanced breeding lines including Ph-5 alone and in combinations with Ph-2 and Ph-3 are being developed. Genetic controls of EB resistance have been studied and advanced breeding lines and cultivars with improved resistance have been developed through traditional breeding. Additionally, QTLs for EB resistance have been identified, which can be utilized for marker-assisted resistance breeding. Currently, new inbred lines and cultivars of tomato with good levels of EB resistance and competitive yield performance are being developed at the Pennsylvania State University. This review will focus on the current knowledge of both LB and EB with respect to the causal pathogens, host resistances, and genetics and breeding progresses.

The adult plant rust resistance loci Lr34/Yr18 and Lr46/Yr29 are important determinants of partial resistance to powdery mildew in bread wheat line Saar

Powdery mildew, caused by Blumeria graminis f. sp. tritici is a major disease of wheat (Triticum aestivum L.) that can be controlled by resistance breeding. The CIMMYT bread wheat line Saar is known for its good level of partial and race non-specific resistance, and the aim of this study was to map QTLs for resistance to powdery mildew in a population of 113 recombinant inbred lines from a cross between Saar and the susceptible line Avocet. The population was tested over 2 years in field trials at two locations in southeastern Norway and once in Beijing, China. SSR markers were screened for association with powdery mildew resistance in a bulked segregant analysis, and linkage maps were created based on selected SSR markers and supplemented with DArT genotyping. The most important QTLs for powdery mildew resistance derived from Saar were located on chromosomes 7DS and 1BL and corresponded to the adult plant rust resistance loci Lr34/Yr18 and Lr46/Yr29. A major QTL was also located on 4BL with resistance contributed by Avocet. Additional QTLs were detected at 3AS and 5AL in the Norwegian testing environments and at 5BS in Beijing. The population was also tested for leaf rust (caused by Puccinia triticina) and stripe rust (caused by P. striiformis f. sp. tritici) resistance and leaf tip necrosis in Mexico. QTLs for these traits were detected on 7DS and 1BL at the same positions as the QTLs for powdery mildew resistance, and confirmed the presence of Lr34/Yr18 and Lr46/Yr29 in Saar. The powdery mildew resistance gene at the Lr34/Yr18 locus has recently been named Pm38. The powdery mildew resistance gene at the Lr46/Yr29 locus is designated as Pm39.

Fine scale genetic and physical mapping using interstitial deletion mutants of Lr34 /Yr18: a disease resistance locus effective against multiple pathogens in wheat

The Lr34/Yr18 locus has contributed to durable, non-race specific resistance against leaf rust (Puccinia triticina) and stripe rust (P. striiformis f. sp. tritici) in wheat (Triticum aestivum). Lr34/Yr18 also cosegregates with resistance to powdery mildew (Pm38) and a leaf tip necrosis phenotype (Ltn1). Using a high resolution mapping family from a cross between near-isogenic lines in the "Thatcher" background we demonstrated that Lr34/Yr18 also cosegregated with stem rust resistance in the field. Lr34/Yr18 probably interacts with unlinked genes to provide enhanced stem rust resistance in "Thatcher". In view of the relatively low levels of DNA polymorphism reported in the Lr34/Yr18 region, gamma irradiation of the single chromosome substitution line, Lalbahadur(Parula7D) that carries Lr34/Yr18 was used to generate several mutant lines. Characterisation of the mutants revealed a range of highly informative genotypes, which included variable size deletions and an overlapping set of interstitial deletions. The mutants enabled a large number of wheat EST derived markers to be mapped and define a relatively small physical region on chromosome 7DS that carried Lr34/Yr18. Fine scale genetic mapping confirmed the physical mapping and identified a genetic interval of less than 0.5 cM, which contained Lr34/Yr18. Both rice and Brachypodium genome sequences provided useful information for fine mapping of ESTs in wheat. Gene order was more conserved between wheat and Brachypodium than with rice but these smaller grass genomes did not reveal sequence information that could be used to identify a candidate gene for rust resistance in wheat. We predict that Lr34/Yr18 is located within a large insertion in wheat not found at syntenic positions in Brachypodium and rice.

Mapping quantitative trait loci controlling common bunt resistance in a doubled haploid population derived from the spring wheat cross RL4452 × AC Domain

Wheat resistance to common bunt is a highly desirable trait for environmentally friendly grain grade protection. Valuable breeding achievements have been made to develop wheat varieties with enhanced resistance to the disease, and mapping of race-specific resistance genes has been reported. However, less is known of the chromosomal regions that control non-race specific resistance to common bunt. In this study, we have characterized a segregating population of 185 doubled haploid spring wheat lines derived from the cross RL4452 × AC Domain. Reactions to a mixture of common bunt races were assessed under field simulated spring-sown conditions in greenhouses in two locations over 2 years. A total 369 polymorphic maker loci including 356 microsatellite loci, five expressed sequences tag (ESTs), and eight genes were used to develop a linkage map. Quantitative trait loci (QTL) analysis using composite interval mapping detected three QTLs associated with common bunt resistance, of which two were located on chromosome 1B and one on chromosome 7A. AC Domain alleles contributed the common bunt resistance at all three QTLs. Usefulness of gene tagging within the identified chromosomal regions for common bunt resistance breeding is discussed.

The epidemiology and management of bacterial blight (Pseudomonas syringae pv. pisi) of field pea (Pisum sativum) in Australia: a review

Bacterial blight caused by Pseudomonas syringae pv. pisi is an important, but sporadic, disease of field peas (Pisum sativum) in Australia. The presence of P. syringae pv. pisi reduces the profitability of peas due to yield loss and, in some cases, it also limits Australia’s export of peas to some countries. Pseudomonoas syringae pv. pisi is primarily a seed-borne pathogen, but infected pea trash can be an important source of inoculum. Alternative hosts and soil are not regarded as epidemiologically important sources of inoculum. P. syringae pv. pisi survives, multiplies and spreads epiphytically in pea crops. Epiphytic populations of P. syringae pv. pisi only become pathogenic following crop damage caused by frost or severe weather conditions. Frost damage is especially important because the ice nucleating activity of P. syringae pv. pisi initiates frost damage at higher temperatures than occurs in the absence of the bacterium. In addition early-sown crops are more prone to damage from bacterial blight than crops sown later in the season. Pseudomonas syringae pv. pisi consists of seven identified races. One of these (Race 6) lacks all avirulence genes and is common around the world and in Australia. Globally, Race 2 and Race 6 predominate; however, in Australia, Race 3 predominates due to the widespread cultivation of cultivars susceptible to Race 3, but resistant to Race 2. Resistance to Race 6 within P. sativum has not been found but attempts are being made to incorporate a race non-specific resistance identified from P. abyssinicum into field pea. Bacterial blight can be successfully controlled using an integrated disease management strategy incorporating crop rotation, pathogen-free seed, avoidance of planting in areas prone to frequent frosts or extreme wet weather, crop hygiene and avoiding early sowing. Seed treatment and application of foliar bactericides have limited use in control of this disease.

Resistance genes to Leptosphaeria maculans in Brassica napus

Black leg of canola (Brassica napus), caused by Leptosphaeria maculans, is primarily controlled by the use of resistant cultivars. In Canada, resistance has been effective for about 10 years, but the frequency of new pathotypes is increasing and there are reports of breakdown of resistance in some cultivars. Race-nonspecific resistance is present in many cultivars in Australia and Europe. The genetic basis of resistance in Canadian cultivars is unknown. This paper reviews our current knowledge relating to disease management using resistance in Canada and the genetics of race-specific and race-nonspecific resistance in the host-pathogen interaction between L. maculans and B. napus. The particular focus is on resistance associated with the A genome and how resistance might be achieved over the long term.

A High-Throughput Gene-Silencing System for the Functional Assessment of Defense-Related Genes in Barley Epidermal Cells

Large-scale gene silencing by RNA interference (RNAi) offers the possibility to address gene function in eukaryotic organisms at a depth unprecedented until recently. Although genome-wide RNAi approaches are being carried out in organisms like Caenorhabditis elegans, Drosophila spp. or human after the corresponding tools had been developed, knock-down of only single or a few genes by RNAi has been reported in plants thus far. Here, we present a method for high-throughput, transient-induced gene silencing (TIGS) by RNAi in barley epidermal cells that is based on biolistic transgene delivery. This method will be useful to address gene function of shoot epidermis resulting in cell-autonomous phenotypes such as resistance or susceptibility to the powdery-mildew fungus Blumeria graminis f. sp. hordei. Gene function in epidermal cell elongation, stomata regulation, or UV resistance might be addressed as well. Libraries of RNAi constructs can be built up by a new, cost-efficient method that combines highly efficient ligation and recombination by the Gateway cloning system. This method allows cloning of any blunt-ended DNA fragment without the need of adaptor sequences. The final RNAi destination vector was found to direct highly efficient RNAi, as reflected by complete knock-down of a cotransformed green fluorescent protein reporter gene as well as by complete phenolcopy of the recessive loss-of-function mlo resistance gene. By using this method, a role of the t-SNARE protein HvSNAP34 in three types of durable, race-nonspecific resistance was observed.

Identification of quantitative trait loci for race-nonspecific resistance to tan spot in wheat

Tan spot, caused by Pyrenophora tritici-repentis (Ptr), is an economically important foliar disease in the major wheat growing areas throughout the world. Multiple races of the pathogen have been characterized based on their ability to cause necrosis and/or chlorosis on differential wheat lines. In this research, we evaluated a population of recombinant inbred lines derived from a cross between the common wheat varieties Grandin and BR34 for reaction to tan spot caused by Ptr races 1-3 and 5. Composite interval mapping revealed QTLs on the short arm of chromosome 1B and the long arm of chromosome 3B that were significantly associated with resistance to all four races. The effects of the two QTLs varied for the different races. The 1B QTL explained from 13% to 29% of the phenotypic variation, whereas the 3B QTL explained from 13% to 41% of the variation. Additional minor QTLs were detected but not associated with resistance to all races. The host-selective toxin Ptr ToxA, which is produced by races 1 and 2, was not a significant factor in the development of disease in this population. The race-nonspecific resistance derived from BR34 may take precedence over the gene-for-gene interaction known to be associated with the wheat-Ptr system.

RNA Interference of Soybean Isoflavone Synthase Genes Leads to Silencing in Tissues Distal to the Transformation Site and to Enhanced Susceptibility to Phytophthora sojae

Isoflavones are thought to play diverse roles in plant-microbe interactions and are also potentially important to human nutrition and medicine. Isoflavone synthase (IFS) is a key enzyme for the formation of the isoflavones. Here, we examined the consequences of RNAi silencing of genes for this enzyme in soybean (Glycine max). Soybean cotyledon tissues were transformed with Agrobacterium rhizogenes carrying an RNAi silencing construct designed to silence expression of both copies of IFS genes. Approximately 50% of emerging roots were transformed with the RNAi construct, and most transformed roots exhibited >95% silencing of isoflavone accumulation. Silencing of IFS was also demonstrated throughout the entire cotyledon (in tissues distal to the transformation site) both by high-performance liquid chromatography analysis of isoflavones and by real-time reverse transcription-PCR. This distal silencing led to a nearly complete suppression of mRNA accumulation for both the IFS1 and IFS2 genes and of isoflavone accumulations induced by wounding or treatment with the cell wall glucan elicitor from Phytophthora sojae. Preformed isoflavone conjugates were not reduced in distal tissues, suggesting little turnover of these stored isoflavone pools. Distal silencing was established within just 5 d of transformation and was highly efficient for a 3- to 4-d period, after which it was no longer apparent in most experiments. Silencing of IFS was effective in at least two genotypes and led to enhanced susceptibility to P. sojae, disrupting both R gene-mediated resistance in roots and nonrace-specific resistance in cotyledon tissues. The soybean cotyledon system, already a model system for defense signal-response and cell-to-cell signaling, may provide a convenient and effective system for functional analysis of plant genes through gene silencing.