SNP assay to detect the ‘Hyuuga’ red-brown lesion resistance gene for Asian soybean rust

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi Syd., has the potential to become a serious threat to soybean, Glycine max L. Merr., production in the USA. A novel rust resistance gene, Rpp?(Hyuuga), from the Japanese soybean cultivar Hyuuga has been identified and mapped to soybean chromosome 6 (Gm06). Our objectives were to fine-map the Rpp?(Hyuuga) gene and develop a high-throughput single nucleotide polymorphism (SNP) assay to detect this ASR resistance gene. The integration of recombination events from two different soybean populations and the ASR reaction data indicates that the Rpp?(Hyuuga) locus is located in a region of approximately 371 kb between STS70887 and STS70923 on chromosome Gm06. A set of 32 ancestral genotypes which is predicted to contain 95% of the alleles present in current elite North American breeding populations and the sources of the previously reported ASR resistance genes (Rpp1, Rpp2, Rpp3, Rpp4, Rpp5, and rpp5) were genotyped with five SNP markers. We developed a SimpleProbe assay based on melting curve analysis for SNP06-44058 which is tighly linked to the Rpp?(Hyuuga) gene. This SNP assay can differentiate plants/lines that are homozygous/homogeneous or heterozygous/heterogeneous for the resistant and susceptible alleles at the Rpp?(Hyuuga) locus.     

Molecular mapping of two loci that confer resistance to Asian rust in soybean

Asian soybean rust (ASR) is caused by the fungal pathogen Phakopsora pachyrhizi Sydow & Sydow. It was first identified in Brazil in 2001 and quickly infected soybean areas in several countries in South America. Primary efforts to combat this disease must involve the development of resistant cultivars. Four distinct genes that confer resistance against ASR have been reported: Rpp1, Rpp2, Rpp3, and Rpp4. However, no cultivar carrying any of those resistance loci has been released. The main objective of this study was to genetically map Rpp2 and Rpp4 resistance genes. Two F(2:3) populations, derived from the crosses between the resistant lines PI 230970 (Rpp2), PI 459025 (Rpp4) and the susceptible cultivar BRS 184, were used in this study. The mapping populations and parental lines were inoculated with a field isolate of P. pachyrhizi and evaluated for lesion type as resistant (RB lesions) or susceptible (TAN lesions). The mapping populations were screened with SSR markers, using the bulk segregant analysis (BSA) to expedite the identification of linked markers. Both resistance genes showed an expected segregation ratio for a dominant trait. This study allowed mapping Rpp2 and Rpp4 loci on the linkage groups J and G, respectively. The associated markers will be of great value on marker assisted selection for this trait.     

Biphasic gene expression changes elicited by Phakopsora pachyrhizi in soybean correlate with fungal penetration and haustoria formation

Inoculation of soybean (Glycine max) plants with Phakopsora pachyrhizi, the causal organism of Asian soybean rust, elicits a biphasic response characterized by a burst of differential gene expression in the first 12 h. A quiescent period occurs from 24 to 48 h after inoculation, in which P. pachyrhizi continues to develop but does not elicit strong host responses, followed by a second phase of intense gene expression. To correlate soybean responses with P. pachyrhizi growth and development, we inoculated the soybean cultivar Ankur (accession PI462312), which carries the Rpp3 resistance gene, with avirulent and virulent isolates of P. pachyrhizi. The avirulent isolate Hawaii 94-1 elicits hypersensitive cell death that limits fungal growth on Ankur and results in an incompatible response, while the virulent isolate Taiwan 80-2 grows extensively, sporulates profusely, and produces a compatible reaction. Inoculated leaves were collected over a 288-h time course for microarray analysis of soybean gene expression and microscopic analysis of P. pachyrhizi growth and development. The first burst in gene expression correlated with appressorium formation and penetration of epidermal cells, while the second burst of gene expression changes followed the onset of haustoria formation in both compatible and incompatible interactions. The proliferation of haustoria coincided with the inhibition of P. pachyrhizi growth in the incompatible interaction or the beginning of accelerated growth in the compatible interaction. The temporal relationships between P. pachyrhizi growth and host responses provide an important context in which to view interacting gene networks that mediate the outcomes of their interactions.     

Molecular mapping of soybean rust resistance in soybean accession PI 561356 and SNP haplotype analysis of the Rpp1 region in diverse germplasm

Soybean rust (SBR), caused by Phakopsora pachyrhizi Sydow, is one of the most economically important and destructive diseases of soybean [Glycine max (L.) Merr.] and the discovery of novel SBR resistance genes is needed because of virulence diversity in the pathogen. The objectives of this research were to map SBR resistance in plant introduction (PI) 561356 and to identify single nucleotide polymorphism (SNP) haplotypes within the region on soybean chromosome 18 where the SBR resistance gene Rpp1 maps. One-hundred F(2:3) lines derived from a cross between PI 561356 and the susceptible experimental line LD02-4485 were genotyped with genetic markers and phenotyped for resistance to P. pachyrhizi isolate ZM01-1. The segregation ratio of reddish brown versus tan lesion type in the population supported that resistance was controlled by a single dominant gene. The gene was mapped to a 1-cM region on soybean chromosome 18 corresponding to the same interval as Rpp1. A haplotype analysis of diverse germplasm across a 213-kb interval that included Rpp1 revealed 21 distinct haplotypes of which 4 were present among 5 SBR resistance sources that have a resistance gene in the Rpp1 region. Four major North American soybean ancestors belong to the same SNP haplotype as PI 561356 and seven belong to the same haplotype as PI 594538A, the Rpp1-b source. There were no North American soybean ancestors belonging to the SNP haplotypes found in PI 200492, the source of Rpp1, or PI 587886 and PI 587880A, additional sources with SBR resistance mapping to the Rpp1 region.     

Characterization of nonhost resistance of Arabidopsis to the Asian soybean rust

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is a devastating disease of soybean. We report the use of the nonhost plant Arabidopsis thaliana to identify the genetic basis of resistance to P. pachyrhizi. Upon attack by P. pachyrhizi, epidermal cells of wild-type Arabidopsis accumulated H2O2, which likely orchestrates the frequently observed epidermal cell death. However, even when epidermal cell death occurred, fungal hyphae grew on and infection was terminated at the mesophyll boundary. These events were associated with expression of PDF1.2, suggesting that P. pachyrhizi, an ostensible biotroph, mimics aspects of a necrotroph. Extensive colonization of the mesophyll occurred in Arabidopsis pen mutants with defective penetration resistance. Although haustoria were found occasionally in mesophyll cells, the successful establishment of biotrophy failed, as evidenced by the cessation of fungal growth. Double mutants affected in either jasmonic acid or salicylic acid signaling in the pen3-1 background revealed the involvement of both pathways in nonhost resistance (NHR) of Arabidopsis to P. pachyrhizi. Interestingly, expression of AtNHL10, a gene that is expressed in tissue undergoing the hypersensitive response, was only triggered in infected pen3-1 mutants. Thus, a suppression of P. pachyrhizi-derived effectors by PEN3 can be inferred. Our results demonstrate that Arabidopsis can be used to study mechanisms of NHR to ASR.     

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.     

A new soybean rust resistance allele from PI 423972 at the <Emphasis Type="Italic">Rpp4</Emphasis> locus

Phakopsora pachyrhizi is a fungal pathogen and the cause of Asian soybean rust. P. pachyrhizi was first detected in the continental USA in 2004 and has since been a threat to the soybean industry. There are six described loci that harbor resistance to P. pachyrhizi (Rpp) genes. The resistance of PI 423972 was previously shown to be within 5 cM of the Rpp4 locus of PI 459025B, yet had differential reactions when challenged with P. pachyrhizi isolates India 1973 and Taiwan 1972. In this study, the resistance of PI 423972 was mapped to a 187.5 kb interval between the SNP markers GSM0543 and GSM0387 on chromosome 18 (51,397,064 to 51,584,617 bp, Glyma.Wm82.a2) that overlaps the interval for Rpp4 and is designated as Rpp4-b. A unique haplotype is described for PI 423972 that separates it from PI 459025B, 32 North American soybean ancestors, and all described sources of Rpp gene resistance.     

Identification of a new soybean rust resistance gene in PI 567102B

Soybean rust (SBR) caused by Phakopsora pachyrhizi Syd. and P. Syd. is one of the most economically important diseases of soybean (Glycine max (L.) Merr.). Durable resistance to P. pachyrhizi is the most effective long-term strategy to control SBR. The objective of this study was to investigate the genetics of resistance to P. pachyrhizi in soybean accession PI 567102B. This accession was previously identified as resistant to SBR in Paraguay and to P. pachyrhizi isolates from seven states in the USA (Alabama, Florida, Georgia, Louisiana, Mississippi, South Carolina, and Texas). Analysis of two independent populations, one in which F(2) phenotypes were inferred from F(2)-derived F(3) (F(2:3)) families and the other in which F(2) plants had phenotypes measured directly, showed that the resistance in PI 567102B was controlled by a single dominant gene. Two different isolates (MS06-1 and LA04-1) at different locations (Stoneville, MS and Ft. Detrick, MD) were used to independently assay the two populations. Linkage analysis of both populations indicated that the resistance locus was located on chromosome 18 (formerly linkage group G), but at a different location than either Rpp1 or Rpp4, which were previously mapped to this linkage group. Therefore, the SBR resistance in PI 567102B appeared to be conditioned by a previously unreported locus, with an underlying single dominant gene inferred. We propose this gene to be designated Rpp6. Incorporating Rpp6 into improved soybean cultivars may have wide benefits as PI 567102B has been shown to provide resistance to P. pachyrhizi isolates from Paraguay and the US.     

Analysis of the wheat and Puccinia triticina (leaf rust) proteomes during a susceptible host-pathogen interaction

Wheat leaf rust is caused by the fungus Puccinia triticina. The genetics of resistance follows the gene-for-gene hypothesis, and thus the presence or absence of a single host resistance gene renders a plant resistant or susceptible to a leaf rust race bearing the corresponding avirulence gene. To investigate some of the changes in the proteomes of both host and pathogen during disease development, a susceptible line of wheat infected with a virulent race of leaf rust were compared to mock-inoculated wheat using 2-DE (with IEF pH 4-8) and MS. Up-regulated protein spots were excised and analyzed by MALDI-QqTOF MS/MS, followed by cross-species protein identification. Where possible MS/MS spectra were matched to homologous proteins in the NCBI database or to fungal ESTs encoding putative proteins. Searching was done using the MASCOT search engine. Remaining unmatched spectra were then sequenced de novo and queried against the NCBInr database using the BLAST and MS BLAST tools. A total of 32 consistently up-regulated proteins were examined from the gels representing the 9-day post-infection proteome in susceptible plants. Of these 7 are host proteins, 22 are fungal proteins of known or hypothetical function and 3 are unknown proteins of putative fungal origin.     

Soybean Resistance to Phakopsora pachyrhizi as Affected by Acibenzolar‐S‐Methyl,Jasmonic Acid and Silicon

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is one of the most important diseases on soybean. At the moment, ASR is managed mainly with fungicides due to the absence of commercial cultivars with resistance to this disease. This study evaluated the effects of acibenzolar‐Smethyl (ASM), jasmonic acid (JA), potassium silicate (PS) and calcium silicate (CS) on soybean resistance to ASR. The ASM, JA and PS were sprayed to leaves 24 h prior to inoculation with P. pachyrhizi. The CS was amended to the soil. The incubation period (time from the inoculation until symptoms development) was longer for plants growing in soil amended with CS or sprayed with ASM in comparison with plants sprayed with water (control). Plants sprayed with ASM had longer latent period (time from the inoculation until signs appearance) in comparison with the control plants. Plants sprayed with PS showed fewer uredia per cm² of leaf in relation to the control plants. The ASM and PS were the most effective treatments in reducing the ASR symptoms in contrast to the JA and CS treatments. The JA served as an inducer of susceptibility to ASR.     

Genetics and mapping of adult plant rust resistance in soybean PI 587886 and PI 587880A

Two soybean accessions, PI 587886 and PI 587880A, previously identified as having resistance to Phakospora pachyrhizi Syd. (soybean rust, SBR) were used to create two populations (POP-1 and POP-2) segregating for SBR resistance. F₂-derived F₃ (F_2:3) families from each population were grown in a naturally SBR-infected field in Paraguay to determine inheritance and map resistance genes. Over 6,000 plants from 178 families in POP-1 and over 5,000 plants from 160 families in POP-2 were evaluated at R5 for lesion type: immune reaction (IR), reddish-brown (RB), or tan (TAN) colored lesions. Based on the lesion type present, each F_2:3 family was rated as resistant, segregating or susceptible and this classification was used to infer the F₂-phenotype and genotype. For both populations, the F₂ segregation ratios fit a 1:2:1 (resistant:segregating:susceptible) ratio expected for a single gene (P > 0.05). The RB lesions occurred almost exclusively in the heterozygous class, indicating incomplete dominance under the conditions of this study. Molecular markers flanking the locations of the known resistance genes were used to map the resistance gene in both populations to the Rpp1 locus. However, evaluation of PI 587886 and PI 587880A against eight P. pachyrhizi isolates indicated that the resistance allele in these two accessions was different from Rpp1. This test also demonstrated that these accessions were resistant to at least one P. pachyrhizi isolate collected in the southern US. This is the first report of using an adult plant field-screen with natural rust pressure to map SBR resistance.     

RNA-Seq analysis of a soybean near-isogenic line carrying bacterial leaf pustule-resistant and -susceptible alleles

Bacterial leaf pustule (BLP) disease is caused by Xanthomonas axonopodis pv. glycines (Xag). To investigate the plant basal defence mechanisms induced in response to Xag, differential gene expression in near-isogenic lines (NILs) of BLP-susceptible and BLP-resistant soybean was analysed by RNA-Seq. Of a total of 46 367 genes that were mapped to soybean genome reference sequences, 1978 and 783 genes were found to be up- and down-regulated, respectively, in the BLP-resistant NIL relative to the BLP-susceptible NIL at 0, 6, and 12h after inoculation (hai). Clustering analysis revealed that these genes could be grouped into 10 clusters with different expression patterns. Functional annotation based on gene ontology (GO) categories was carried out. Among the putative soybean defence response genes identified (GO:0006952), 134 exhibited significant differences in expression between the BLP-resistant and -susceptible NILs. In particular, pathogen-associated molecular pattern (PAMP) and damage-associated molecular pattern (DAMP) receptors and the genes induced by these receptors were highly expressed at 0 hai in the BLP-resistant NIL. Additionally, pathogenesis-related (PR)-1 and -14 were highly expressed at 0 hai, and PR-3, -6, and -12 were highly expressed at 12 hai. There were also significant differences in the expression of the core JA-signalling components MYC2 and JASMONATE ZIM-motif. These results indicate that powerful basal defence mechanisms involved in the recognition of PAMPs or DAMPs and a high level of accumulation of defence-related gene products may contribute to BLP resistance in soybean.