Allelic variability in the Rpp1 locus conferring resistance to Asian soybean rust revealed by genome-wide association.

Soybean is a crucial crop for the Brazilian economy, but it faces challenges from the biotrophic fungus Phakopsora pachyrhizi, which causes Asian Soybean Rust (ASR). In this study, we aimed to identify SNPs associated with resistance within the Rpp1 locus, which is effective against Brazilian ASR populations. We employed GWAS and re-sequencing analyzes to pinpoint SNP markers capable of differentiating between soybean accessions harboring the Rpp1, Rpp1-b and other alternative alleles in the Rpp1 locus and from susceptible soybean cultivars. Seven SNP markers were found to be associated with ASR resistance through GWAS, with three of them defining haplotypes that efficiently distinguished the accessions based on their ASR resistance and source of the Rpp gene. These haplotypes were subsequently validated using a bi-parental population and a diverse set of Rpp sources, demonstrating that the GWAS markers co-segregate with ASR resistance. We then examined the presence of these haplotypes in a diverse set of soybean genomes worldwide, finding a few new potential sources of Rpp1/Rpp1-b. Further genomic sequence analysis revealed nucleotide differences within the genes present in the Rpp1 locus, including the ULP1-NBS-LRR genes, which are potential R gene candidates. These results provide valuable insights into ASR resistance in soybean, thus helping the development of resistant soybean varieties through genetic breeding programs.

Identification and characterization of a new soybean promoter induced by Phakopsora pachyrhizi, the causal agent of Asian soybean rust.

BACKGROUND: Phakopsora pachyrhizi is a biotrophic fungal pathogen responsible for the Asian soybean rust disease causing important yield losses in tropical and subtropical soybean-producing countries. P. pachyrhizi triggers important transcriptional changes in soybean plants during infection, with several hundreds of genes being either up- or downregulated. RESULTS: Based on published transcriptomic data, we identified a predicted chitinase gene, referred to as GmCHIT1, that was upregulated in the first hours of infection. We first confirmed this early induction and showed that this gene was expressed as early as 8 h after P. pachyrhizi inoculation. To investigate the promoter of GmCHIT1, transgenic soybean plants expressing the green fluorescence protein (GFP) under the control of the GmCHIT1 promoter were generated. Following inoculation of these transgenic plants with P. pachyrhizi, GFP fluorescence was detected in a limited area located around appressoria, the fungal penetration structures. Fluorescence was also observed after mechanical wounding whereas no variation in fluorescence of pGmCHIT1:GFP transgenic plants was detected after a treatment with an ethylene precursor or a methyl jasmonate analogue. CONCLUSION: We identified a soybean chitinase promoter exhibiting an early induction by P. pachyrhizi located in the first infected soybean leaf cells. Our results on the induction of GmCHIT1 promoter by P. pachyrhizi contribute to the identification of a new pathogen inducible promoter in soybean and beyond to the development of a strategy for the Asian soybean rust disease control using biotechnological approaches.

The Arabidopsis non-host defence-associated coumarin scopoletin protects soybean from Asian soybean rust.

The fungus Phakopsora pachyrhizi (Pp) causes Asian soybean rust (SBR) disease which provokes tremendous losses in global soybean production. Pp is mainly controlled with synthetic fungicides to which the fungus swiftly develops fungicide resistance. To substitute or complement synthetic fungicides in Asian soybean rust control, we aimed to identify antifungal metabolites in Arabidopsis which is not a host for Pp. Comparative transcriptional and metabolic profiling of the Pp-inoculated Arabidopsis non-host and the soybean host revealed induction of phenylpropanoid metabolism-associated genes in both species but activation of scopoletin biosynthesis only in the resistant non-host. Scopoletin is a coumarin and an antioxidant. In vitro experiments disclosed fungistatic activity of scopoletin against Pp, associated with reduced accumulation of reactive oxygen species (ROS) in fungal pre-infection structures. Non-antioxidant and antioxidant molecules including coumarins with a similar structure to scopoletin were inactive or much less effective at inhibiting fungal accumulation of ROS and germination of Pp spores. When sprayed onto Arabidopsis leaves, scopoletin also suppressed the formation of Pp pre-infection structures and penetration of the plant. However, scopoletin neither directly activated defence nor did it prime Arabidopsis for enhanced defence, therefore emphasizing fungistatic activity as the exclusive mode of action of scopoletin against Pp. Because scopletin also protected soybean from Pp infection, the coumarin may serve as a natural fungicide or as a lead for the development of near-to-nature fungicides against Asian soybean rust.

Rpp1 Encodes a ULP1-NBS-LRR Protein That Controls Immunity to Phakopsora pachyrhizi in Soybean.

Phakopsora pachyrhizi is the causal agent of Asian soybean rust. Susceptible soybean plants infected by virulent isolates of P. pachyrhizi are characterized by tan-colored lesions and erumpent uredinia on the leaf surface. Germplasm screening and genetic analyses have led to the identification of seven loci, Rpp1 to Rpp7, that provide varying degrees of resistance to P. pachyrhizi (Rpp). Two genes, Rpp1 and Rpp1b, map to the same region on soybean chromosome 18. Rpp1 is unique among the Rpp genes in that it confers an immune response (IR) to avirulent P. pachyrhizi isolates. The IR is characterized by a lack of visible symptoms, whereas resistance provided by Rpp1b to Rpp7 results in red-brown foliar lesions. Rpp1 maps to a region spanning approximately 150 kb on chromosome 18 between markers Sct_187 and Sat_064 in L85-2378 (Rpp1), an isoline developed from Williams 82 and PI 200492 (Rpp1). To identify Rpp1, we constructed a bacterial artificial chromosome library from soybean accession PI 200492. Sequencing of the Rpp1 locus identified three homologous nucleotide binding site-leucine rich repeat (NBS-LRR) candidate resistance genes between Sct_187 and Sat_064. Each candidate gene is also predicted to encode an N-terminal ubiquitin-like protease 1 (ULP1) domain. Cosilencing of the Rpp1 candidates abrogated the immune response in the Rpp1 resistant soybean accession PI 200492, indicating that Rpp1 is a ULP1-NBS-LRR protein and plays a key role in the IR.

Effect of Selected Biopesticides in Reducing Soybean Rust (Phakopsora pachyrhizi) Development.

The intensive use of fungicides in controlling soybean rust (SBR), a damaging foliar fungal disease of soybean caused by the obligate fungus Phakopsora pachyrhizi, may have accelerated the insensitivity of P. pachyrhizi populations to fungicides. The objective of this study was to determine the effect of selected biopesticides and their application time on reducing SBR infection. There were differences (P < 0.05) in percent rust reduction values for application times, biopesticide treatments, and their interaction in detached-leaf and whole-plant greenhouse experiments. All application times and nearly all biopesticide treatments reduced (α = 0.05) fungal infection compared with the nonfungicide control. Among the treatments, Bacillus subtilis QST 713 and acibenzolar-S-methyl often reduced fungal sporulation more than the other treatments in detached-leaf and whole-plant greenhouse experiments. The identification of biopesticides effective to P. pachyrhizi may be a valuable alternative or complement to synthetic fungicides and may be useful in integrated pest management programs for SBR control.

Relationship between biochemical and photosynthetic traits with Asian soybean rust.

Asian soybean rust (Phakopsora pachyrhizi - ASR) is one of the major diseases that occur in soybean and causes great damage to commercial crops. Therefore, the goal of this work was to investigate the relationship between biochemical and photosyntetic parameters in soybean with ASR. Two experiments were performed in a randomized complete block with three treatments (water, Tween 20, and methyl jasmonate). The evaluated traits were: severity, chlorophyll pools, concentration of phenolic compounds content, enzyme activity, and photosyntetic parameters. Based on Pearson correlation, correlation network and path analysis it was verified that the severity had high correlation with almost all traits evaluated meanwhile photosynthesis is weakly related with almost all traits. Therefore, the occurrence of ASR affects directly the traits related to enzymatic activity and phenolic compounds content in soybean plants inoculated with ASR. The management of ASR is important to keep in normal levels the rates of photosynthesis carried out by the plant, and thus not affect the yield. Besides that, understanding the biochemical mechanisms and ecophysiological responses that occur during the soybean-P. pachyrhizi interaction has a great importance for breeding programs, as it will help for obtaining resistant cultivars or efficient methods in fungus control.

Prediction of Short-Distance Aerial Movement of Phakopsora pachyrhizi Urediniospores Using Machine Learning.

Dispersal of urediniospores by wind is the primary means of spread for Phakopsora pachyrhizi, the cause of soybean rust. Our research focused on the short-distance movement of urediniospores from within the soybean canopy and up to 61 m from field-grown rust-infected soybean plants. Environmental variables were used to develop and compare models including the least absolute shrinkage and selection operator regression, zero-inflated Poisson/regular Poisson regression, random forest, and neural network to describe deposition of urediniospores collected in passive and active traps. All four models identified distance of trap from source, humidity, temperature, wind direction, and wind speed as the five most important variables influencing short-distance movement of urediniospores. The random forest model provided the best predictions, explaining 76.1 and 86.8% of the total variation in the passive- and active-trap datasets, respectively. The prediction accuracy based on the correlation coefficient (r) between predicted values and the true values were 0.83 (P < 0.0001) and 0.94 (P < 0.0001) for the passive and active trap datasets, respectively. Overall, multiple machine learning techniques identified the most important variables to make the most accurate predictions of movement of P. pachyrhizi urediniospores short-distance.

ABC transporter PEN3/PDR8/ABCG36 interacts with calmodulin that, like PEN3, is required for Arabidopsis nonhost resistance.

Nonhost resistance (NHR) is the most prevalent form of plant immunity. In Arabidopsis, NHR requires membrane-localized ATP-binding cassette (ABC) transporter PENETRATION (PEN) 3. Upon perception of pathogen-associated molecular patterns, PEN3 becomes phosphorylated, suggestive of PEN3 regulation by post-translational modification. Here, we investigated the PEN3 protein interaction network. We probed the Arabidopsis protein microarray AtPMA-5000 with the N-terminal cytoplasmic domain of PEN3. Several of the proteins identified to interact with PEN3 in vitro represent cellular Ca(2+) sensors, including calmodulin (CaM) 3, CaM7 and several CaM-like proteins, pointing to the importance of Ca(2+) sensing to PEN3-mediated NHR. We demonstrated co-localization of PEN3 and CaM7, and we confirmed PEN3-CaM interaction in vitro and in vivo by PEN3 pull-down with CaM Sepharose, CaM overlay assay and bimolecular fluorescence complementation. We also show that just like in pen3, NHR to the nonadapted fungal pathogens Phakopsora pachyrhizi and Blumeria graminis f.sp. hordei is compromised in the Arabidopsis cam7 and pen3 cam7 mutants. Our study discloses CaM7 as a PEN3-interacting protein crucial to Arabidopsis NHR and emphasizes the importance of Ca(2+) sensing to plant immunity.

Competitive Fitness of Phakopsora pachyrhizi Isolates with Mutations in the CYP51 and CYTB Genes.

Soybean rust (Phakopsora pachyrhizi) in Brazil is mainly controlled with applications of fungicides, including demethylation inhibitors (DMI) and quinone outside inhibitors (QoI). Isolates with less sensitivity to DMI and QoI have been reported, and these have been found to have mutations in the CYP51 and CYTB genes, respectively. There have been no reports of fitness costs in isolates with mutations in CYP51 and CYTB, and the aim of this work was to compare the competitive ability of isolates with lower DMI or QoI sensitivities with that of sensitive (wild-type) isolates. Urediniospores of sensitive wild-type isolates and isolates with different CYP51 or CYTB alleles were mixed and inoculated on detached soybean leaves. After 3 weeks, urediniospores were harvested and used as inoculum for the next disease cycle. Frequencies of relevant target site mutations were monitored using the pyrosequencing method over four disease cycles. Isolates with lower DMI sensitivity and different CYP51 alleles had competitive disadvantages compared with a DMI-sensitive, wild-type CYP51 isolate. In contrast, the isolate with the F129L mutation in the CYTB gene competed equally well with a QoI-sensitive, wild-type CYTB isolate under the conditions of this experiment. The CYP51 and CYTB alleles were stable in all isolates over four disease cycles when cultivated alone.

History, Epidemic Evolution, and Model Burn-In for a Network of Annual Invasion: Soybean Rust.

Ecological history may be an important driver of epidemics and disease emergence. We evaluated the role of history and two related concepts, the evolution of epidemics and the burn-in period required for fitting a model to epidemic observations, for the U.S. soybean rust epidemic (caused by Phakopsora pachyrhizi). This disease allows evaluation of replicate epidemics because the pathogen reinvades the United States each year. We used a new maximum likelihood estimation approach for fitting the network model based on observed U.S. epidemics. We evaluated the model burn-in period by comparing model fit based on each combination of other years of observation. When the miss error rates were weighted by 0.9 and false alarm error rates by 0.1, the mean error rate did decline, for most years, as more years were used to construct models. Models based on observations in years closer in time to the season being estimated gave lower miss error rates for later epidemic years. The weighted mean error rate was lower in backcasting than in forecasting, reflecting how the epidemic had evolved. Ongoing epidemic evolution, and potential model failure, can occur because of changes in climate, host resistance and spatial patterns, or pathogen evolution.

From Select Agent to an Established Pathogen: The Response to Phakopsora pachyrhizi (Soybean Rust) in North America.

The pathogen causing soybean rust, Phakopsora pachyrhizi, was first described in Japan in 1902. The disease was important in the Eastern Hemisphere for many decades before the fungus was reported in Hawaii in 1994, which was followed by reports from countries in Africa and South America. In 2004, P. pachyrhizi was confirmed in Louisiana, making it the first report in the continental United States. Based on yield losses from countries in Asia, Africa, and South America, it was clear that this pathogen could have a major economic impact on the yield of 30 million ha of soybean in the United States. The response by agencies within the United States Department of Agriculture, industry, soybean check-off boards, and universities was immediate and complex. The impacts of some of these activities are detailed in this review. The net result has been that the once dreaded disease, which caused substantial losses in other parts of the world, is now better understood and effectively managed in the United States. The disease continues to be monitored yearly for changes in spatial and temporal distribution so that soybean growers can continue to benefit by knowing where soybean rust is occurring during the growing season.

Reference Genes in the Pathosystem Phakopsora pachyrhizi/ Soybean Suitable for Normalization in Transcript Profiling.

Phakopsora pachyrhizi is a devastating pathogen on soybean, endangering soybean production worldwide. Use of Host Induced Gene Silencing (HIGS) and the study of effector proteins could provide novel strategies for pathogen control. For both approaches quantification of transcript abundance by RT-qPCR is essential. Suitable stable reference genes for normalization are indispensable to obtain accurate RT-qPCR results. According to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and using algorithms geNorm and NormFinder we tested candidate reference genes from P. pachyrhizi and Glycine max for their suitability in normalization of transcript levels throughout the infection process. For P. pachyrhizi we recommend a combination of CytB and PDK or GAPDH for in planta experiments. Gene expression during in vitro stages and over the whole infection process was found to be highly unstable. Here, RPS14 and UbcE2 are ranked best by geNorm and NormFinder. Alternatively CytB that has the smallest Cq range (Cq: quantification cycle) could be used. We recommend specification of gene expression relative to the germ tube stage rather than to the resting urediospore stage. For studies omitting the resting spore and the appressorium stages a combination of Elf3 and RPS9, or PKD and GAPDH should be used. For normalization of soybean genes during rust infection Ukn2 and cons7 are recommended.

Integration of genome-wide association studies and gene coexpression networks unveils promising soybean resistance genes against five common fungal pathogens.

Soybean is one of the most important legume crops worldwide. However, soybean yield is dramatically affected by fungal diseases, leading to economic losses of billions of dollars yearly. Here, we integrated publicly available genome-wide association studies and transcriptomic data to prioritize candidate genes associated with resistance to Cadophora gregata, Fusarium graminearum, Fusarium virguliforme, Macrophomina phaseolina, and Phakopsora pachyrhizi. We identified 188, 56, 11, 8, and 3 high-confidence candidates for resistance to F. virguliforme, F. graminearum, C. gregata, M. phaseolina and P. pachyrhizi, respectively. The prioritized candidate genes are highly conserved in the pangenome of cultivated soybeans and are heavily biased towards fungal species-specific defense responses. The vast majority of the prioritized candidate resistance genes are related to plant immunity processes, such as recognition, signaling, oxidative stress, systemic acquired resistance, and physical defense. Based on the number of resistance alleles, we selected the five most resistant accessions against each fungal species in the soybean USDA germplasm. Interestingly, the most resistant accessions do not reach the maximum theoretical resistance potential. Hence, they can be further improved to increase resistance in breeding programs or through genetic engineering. Finally, the coexpression network generated here is available in a user-friendly web application ( https://soyfungigcn.venanciogroup.uenf.br/ ) and an R/Shiny package ( https://github.com/almeidasilvaf/SoyFungiGCN ) that serve as a public resource to explore soybean-pathogenic fungi interactions at the transcriptional level.

Unraveling Asian Soybean Rust metabolomics using mass spectrometry and Molecular Networking approach.

Asian Soybean Rust (ASR), caused by the biotrophic fungus Phakopsora pachyrhizi, is a devastating disease with an estimated crop yield loss of up to 90%. Yet, there is a nerf of information on the metabolic response of soybean plants to the pathogen Untargeted metabolomics and Global Natural Products Social Molecular Networking platform approach was used to explore soybean metabolome modulation to P. pachyrhizi infection. Soybean plants susceptible to ASR was inoculated with P. pachyrhizi spore suspension and non-inoculated plants were used as controls. Leaves from both groups were collected 14 days post-inoculation and extracted using different extractor solvent mixtures. The extracts were analyzed on an ultra-high performance liquid chromatography system coupled to high-definition electrospray ionization-mass spectrometry. There was a significant production of defense secondary metabolites (phenylpropanoids, terpenoids and flavonoids) when P. pachyrhizi infected soybean plants, such as putatively identified liquiritigenin, coumestrol, formononetin, pisatin, medicarpin, biochanin A, glyoceollidin I, glyoceollidin II, glyoceollin I, glyoceolidin II, glyoceolidin III, glyoceolidin IV, glyoceolidin VI. Primary metabolites (amino acids, peptides and lipids) also were putatively identified. This is the first report using untargeted metabolomics and GNPS-Molecular Networking approach to explore ASR in soybean plants. Our data provide insights into the potential role of some metabolites in the plant resistance to ASR, which could result in the development of resistant genotypes of soybean to P. pachyrhizi, and effective and specific products against the pathogen.

Reduction of Phakopsora pachyrhizi infection on soybean through host- and spray-induced gene silencing.

Asian soybean rust (ASR), caused by the obligate fungal pathogen Phakopsora pachyrhizi, often leads to significant yield losses and can only be managed through fungicide applications currently. In the present study, eight urediniospore germination or appressorium formation induced P. pachyrhizi genes were investigated for their feasibility to suppress ASR through a bean pod mottle virus (BPMV)-based host-induced gene silencing (HIGS) strategy. Soybean plants expressing three of these modified BPMV vectors suppressed the expression of their corresponding target gene by 45%-80%, fungal biomass accumulation by 58%-80%, and significantly reduced ASR symptom development in soybean leaves after the plants were inoculated with P. pachyrhizi, demonstrating that HIGS can be used to manage ASR. In addition, when the in vitro synthesized double-stranded RNAs (dsRNAs) for three of the genes encoding an acetyl-CoA acyltransferase, a 40S ribosomal protein S16, and glycine cleavage system H protein were sprayed directly onto detached soybean leaves prior to P. pachyrhizi inoculation, they also resulted in an average of over 73% reduction of pustule numbers and 75% reduction in P. pachyrhizi biomass accumulation on the detached leaves compared to the controls. To the best of our knowledge, this is the first report of suppressing P. pachyrhizi infection in soybean through both HIGS and spray-induced gene silencing. It was demonstrated that either HIGS constructs targeting P. pachyrhizi genes or direct dsRNA spray application could be an effective strategy for reducing ASR development on soybean.

Phakopsora pachyrhizi triggers the jasmonate signaling pathway during compatible interaction in soybean and GmbZIP89 plays a role of major component in the pathway.

The biotrophic fungus Phakopsora pachyrhizi is currently the major pathogen affecting soybean production worldwide. It has already been suggested for the non-host interaction between P. pachyrhizi and Arabidopsis thaliana that the fungus in early infection induces jasmonic acid (JA) pathway to the detriment of the salicylic acid (SA) pathway as a mechanism to the establishment of infection. In this study, we verified that this mechanism might also be occurring during the compatible interaction in soybean (Glycine max L. Merril). It was demonstrated that P. pachyrhizi triggers a JA pathway during the early and late stages of infection in a susceptible soybean cultivar. The expression of the GmbZIP89 was induced in a biphasic profile, similarly to other JA responsive genes, which indicates a new marker gene for this signaling pathway. Additionally, plants silenced for GmbZIP89 (iGmZIP89) by the virus-induced gene silencing (VIGS) approach present lower severity of infection and higher expression of pathogenesis related protein 1 (PR1). The lower disease severity showed that the iGmbZIP89 plants became more resistant to infection. These data corroborate the hypothesis that the GmbZIP89 may be a resistance negative regulator. In conclusion, we demonstrated that P. pachyrhizi mimics a necrotrophic fungus and activates the JA/ET pathway in soybean. It is possible to suppose that its direct penetration on epidermal cells or fungal effectors may modulate the expression of target genes aiming the activation of the JA pathway and inhibition of SA defense.

Soybean leaves transcriptomic data dissects the phenylpropanoid pathway genes as a defence response against Phakopsora pachyrhizi.

Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, is responsible for severe yield losses of up to 90% in all soybean producing countries. Till today, eight resistance to Phakopsora pachyrhizi (Rpp) loci have been mapped in soybean. Their resistance mechanism is race specific but largely unknown. The transcriptomes of susceptible BRS184 and Rpp3 with ASR isolates T1-2 at 24 h after inoculation (hai) and without ASR inoculation (mock) were annotated by similarity searching with different databases. A total of 4518 differentially expressed genes were identified. We found 70.89%, 56.61%, 32.13%, and 56.04% genes in the protein family databases (PFAM), Gene Ontology (GO), Eukaryotic clusters of Orthologous Groups (KOG), and Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG), respectively. KEGG disclosed that 52% of the phenylpropanoid pathway related genes were up-regulated. The relative gene expression study for selected genes of that pathway was conducted by RT-qPCR using Rpp1-Rpp4 carrying lines with T1-2 infection. The RT-qPCR results revealed that the Rpp lines utilized these genes in a rate limiting manner as a defence response. With the exception of glycinol 4-dimethylallyltransferase (G4DT) and chalcone reductase (CHR), all the genes showed the greatest expression at 12 hai, but the gene expressions which occur between 24 and 96 hai make these Rpp lines unique to their respective ASR isolates. Moreover, functional coordination of arogenate dehydratase 6 (ADT6) and 4-hydroxy-3-methylbut-2-enyl diphosphate synthase (ispG), chalcone synthase (CHS) and CHR, and G4DT and phytyltransferase 3 (PT3) may have a great impact on soybean resistance against ASR.

A pigeonpea gene confers resistance to Asian soybean rust in soybean.

Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, is one of the most economically important crop diseases, but is only treatable with fungicides, which are becoming less effective owing to the emergence of fungicide resistance. There are no commercial soybean cultivars with durable resistance to P. pachyrhizi, and although soybean resistance loci have been mapped, no resistance genes have been cloned. We report the cloning of a P. pachyrhizi resistance gene CcRpp1 (Cajanus cajan Resistance against Phakopsora pachyrhizi 1) from pigeonpea (Cajanus cajan) and show that CcRpp1 confers full resistance to P. pachyrhizi in soybean. Our findings show that legume species related to soybean such as pigeonpea, cowpea, common bean and others could provide a valuable and diverse pool of resistance traits for crop improvement.

Molecular Characterization of Resistance to Soybean Rust (Phakopsora pachyrhizi Syd. & Syd.) in Soybean Cultivar DT 2000 (PI 635999).

Resistance to soybean rust (SBR), caused by Phakopsora pachyrhizi Syd. & Syd., has been identified in many soybean germplasm accessions and is conferred by either dominant or recessive genes that have been mapped to six independent loci (Rpp1 -Rpp6), but No U.S. cultivars are resistant to SBR. The cultivar DT 2000 (PI 635999) has resistance to P. pachyrhizi isolates and field populations from the United States as well as Vietnam. A F6:7 recombinant inbred line (RIL) population derived from Williams 82 × DT 2000 was used to identify genomic regions associated with resistance to SBR in the field in Ha Noi, Vietnam, and in Quincy, Florida, in 2008. Bulked segregant analysis (BSA) was conducted using the soybean single nucleotide polymorphism (SNP) USLP 1.0 panel along with simple sequence repeat (SSR) markers to detect regions of the genome associated with resistance. BSA identified four BARC_SNP markers near the Rpp3 locus on chromosome (Chr.) 6. Genetic analysis identified an additional genomic region around the Rpp4 locus on Chr. 18 that was significantly associated with variation in the area under disease progress curve (AUDPC) values and sporulation in Vietnam. Molecular markers tightly linked to the DT 2000 resistance alleles on Chrs. 6 and 18 will be useful for marker-assisted selection and backcrossing in order to pyramid these genes with other available SBR resistance genes to develop new varieties with enhanced and durable resistance to SBR.

Transcriptomic and metabolomic analyses identify a role for chlorophyll catabolism and phytoalexin during Medicago nonhost resistance against Asian soybean rust.

Asian soybean rust (ASR) caused by Phakopsora pachyrhizi is a devastating foliar disease affecting soybean production worldwide. Understanding nonhost resistance against ASR may provide an avenue to engineer soybean to confer durable resistance against ASR. We characterized a Medicago truncatula-ASR pathosystem to study molecular mechanisms of nonhost resistance. Although urediniospores formed appressoria and penetrated into epidermal cells of M. truncatula, P. pachyrhizi failed to sporulate. Transcriptomic analysis revealed the induction of phenylpropanoid, flavonoid and isoflavonoid metabolic pathway genes involved in the production of phytoalexin medicarpin in M. truncatula upon infection with P. pachyrhizi. Furthermore, genes involved in chlorophyll catabolism were induced during nonhost resistance. We further characterized one of the chlorophyll catabolism genes, Stay-green (SGR), and demonstrated that the M. truncatula sgr mutant and alfalfa SGR-RNAi lines showed hypersensitive-response-like enhanced cell death upon inoculation with P. pachyrhizi. Consistent with transcriptomic analysis, metabolomic analysis also revealed the accumulation of medicarpin and its intermediate metabolites. In vitro assay showed that medicarpin inhibited urediniospore germination and differentiation. In addition, several triterpenoid saponin glycosides accumulated in M. truncatula upon inoculation with P. pachyrhizi. In summary, using multi-omic approaches, we identified a correlation between phytoalexin production and M. truncatula defense responses against ASR.