Identification of the soybean HyPRP family and specific gene response to Asian soybean rust disease

Soybean [Glycine max (L.) Merril], one of the most important crop species in the world, is very susceptible to abiotic and biotic stress. Soybean plants have developed a variety of molecular mechanisms that help them survive stressful conditions. Hybrid proline-rich proteins (HyPRPs) constitute a family of cell-wall proteins with a variable N-terminal domain and conserved C-terminal domain that is phylogenetically related to non-specific lipid transfer proteins. Members of the HyPRP family are involved in basic cellular processes and their expression and activity are modulated by environmental factors. In this study, microarray analysis and real time RT-qPCR were used to identify putative HyPRP genes in the soybean genome and to assess their expression in different plant tissues. Some of the genes were also analyzed by time-course real time RT-qPCR in response to infection by Phakopsora pachyrhizi, the causal agent of Asian soybean rust disease. Our findings indicate that the time of induction of a defense pathway is crucial in triggering the soybean resistance response to P. pachyrhizi. This is the first study to identify the soybean HyPRP group B family and to analyze disease-responsive GmHyPRP during infection by P. pachyrhizi.     

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.     

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.     

Phakopsora pachyrhizi, the causal agent of Asian soybean rust

The plant pathogenic basidiomycete fungi Phakopsora pachyrhizi and Phakopsora meibomiae cause rust disease in soybean plants. Phakopsora pachyrhizi originated in Asia–Australia, whereas the less aggressive P. meibomiae originated in Latin America. In the New World, P. pachyrhizi was first reported in the 1990s to have spread to Hawaii and, since 2001, it has been found in South America. In 2004, the pathogen entered continental USA. This review provides detailed information on the taxonomy and molecular biology of the pathogen, and summarizes strategies to combat the threat of this devastating disease.
Taxonomy: Phakopsora pachyrhizi Syd. & P. Syd; uredial anamorph: Malupa sojae (syn. Uredo sojae ); Domain Eukaryota; Kingdom Fungi; Phylum Basidiomycota; Order Uredinales; Class Urediniomycetes; Family Phakopsoraceae; Genus Phakopsora ( http://www.indexfungorum.org ). The nomenclature of rust spores and spore-producing structures used within this review follows Agrios GN (2005) Plant Pathology , 5th edn. London: Elsevier/Academic Press.
Host range: In the field, P. pachyrhizi infects leaf tissue from a broad range (at least 31 species in 17 genera) of leguminous plants. Infection of an additional 60 species in other genera has been achieved under laboratory conditions.
Disease symptoms: At the beginning of the disease, small, tan-coloured lesions, restricted by leaf veins, can be observed on infected soybean leaves. Lesions enlarge and, 5–8 days after initial infection, rust pustules (uredia, syn. uredinia) become visible. Uredia develop more frequently in lesions on the lower surface of the leaf than on the upper surface. The uredia open with a round ostiole through which uredospores are released.     

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.     

Proteomic analysis of germinating urediniospores of Phakopsora pachyrhizi,causal agent of Asian soybean rust

Phakopsora pachyrhizi is an obligate pathogen that causes Asian soybean rust. Asian soybean rust has an unusually broad host range and infects by direct penetration through the leaf cuticle. In order to understand the early events in the infection process, it is important to identify and characterize proteins in P. pachyrhizi. Germination of the urediniospore is the first stage in the infection process and represents a critical life stage applicable to studies with this obligate pathogen. We have applied a 2‐DE and MS approach to identify 117 proteins from the National Center of Biotechnology Information nonredundant protein database and a custom database of Basidiomycota EST sequences. Proteins with roles in primary metabolism, energy transduction, stress, cellular regulation and signaling were identified in this study. This data set is accessible at http://world‐2dpage.expasy.org/repository/database=0018 .     

Molecular characterization of beta-tubulin from Phakopsora pachyrhizi, the causal agent of Asian soybean rust

β-tubulins are structural components of microtubules and the targets of benzimidazole fungicides used to control many diseases of agricultural importance. Intron polymorphisms in the intron-rich genes of these proteins have been used in phylogeographic investigations of phytopathogenic fungi. In this work, we sequenced 2764 nucleotides of the β-tubulin gene (Pp tubB) in samples of Phakopsora pachyrhizi collected from seven soybean fields in Brazil. Pp tubB contained an open reading frame of 1341 nucleotides, including nine exons and eight introns. Exon length varied from 14 to 880 nucleotides, whereas intron length varied from 76 to 102 nucleotides. The presence of only four polymorphic sites limited the usefulness of Pp tubB for phylogeographic studies in P. pachyrhizi. The gene structures of Pp tubB and orthologous β-tubulin genes of Melampsora lini and Uromyces viciae-fabae were highly conserved. The amino acid substitutions in β-tubulin proteins associated with the onset of benzimidazole resistance in model organisms, especially at His (6) , Glu (198) and Phe (200) , were absent from the predicted sequence of the P. pachyrhizi β-tubulin protein.     

Functional dietary response of Asian black bears to changes in sika deer density

Omnivores are generally opportunistic foragers and have a flexible dietary response to resource abundance and availability. Their populations may consist of individuals that differ from each other in terms of their trophic positions, which implies that the dietary response to resource fluctuations differs within a population. We investigated how changes in the abundance of sika deer (Cervus nippon) affected dietary variation and body condition in the Asian black bear (Ursus thibetanus). We used fecal analysis, nitrogen stable isotopes (δ¹⁵N), and body measurements to determine whether the variation in dietary meat content of Asian black bears is positively related to variations in the density of the sika deer population, whether male bears have a higher trophic position compared to females, and whether dietary meat content is positively related with body mass or body condition of bears. We found a positive correlation between the occurrence of deer remains in bear feces and deer density, suggesting that bears change their diet in response to temporal changes in deer density. Male bears had higher δ¹⁵N values than females, and neither values varied when deer density decreased. Males selectively consumed deer after a reduction in deer density, whereas females consistently consumed more plant-based diet. The δ¹⁵N values were positively related with body mass of adult (>4 yr) bears but had no relationship with body condition of bears of either sex or any age class. Deer seem to be an important food source for large adult males, which have an advantage in mating. Thus, increasing herbivore abundance and availability altered the foraging strategy of Asian black bears, but the importance of herbivore on bear diet differs within a population.     

Dynamic reorganization of microtubules and microfilaments in flax cells during the resistance response to flax rust infection

The cytoskeleton in plant cells is a dynamic structure that can rapidly respond to extracellular stimuli. Alteration of the organization of microtubules and actin microfilaments was examined in mesophyll cells of flax, Linum usitatissimum L., during attempted infection by the flax rust fungus, Melampsora lini (Ehrenb.) Lev. Flax leaves that had been inoculated with either a compatible (yielding a susceptible reaction) or an incompatible (yielding a resistant reaction) strain of M. lini were embedded in butyl-methylmethacrylate resin; sections of this material were immunofluorescently labelled with anti-tubulin or anti-actin and examined using confocal laser scanning microscopy. In uninfected leaves, microtubules in the mesophyll cells formed a transverse array in the cell cortex. Microfilaments radiated through the cytoplasm from the nucleus. In an incompatible interaction, microtubules and microfilaments were extensively reorganized in mesophyll cells that were in contact with fungal infection hyphae or haustorial mother cells before penetration of the cell by the infection peg. After the initiation of haustorium development, microtubules disappeared from the infected cells, and growth of the haustoria ceased. In an incompatible interaction, hypersensitive cell death occurred in more than 70% of infected cells but occurred in less than 20% of cells in compatible interactions. After the infected cell had undergone hypersensitive cell death, the cytoskeleton in neighbouring cells became focused on the walls shared with the necrotic cell. In compatible interactions, reorganization of the cytoskeleton was either not observed at all or was observed much less frequently up to 48 h after inoculation.Abbreviations FITC fluorescein isothiocyanate - WGA wheatgerm agglutinin We thank Dr. G.J. Lawrence for providing valuable discussions and materials.     

Ultrastructural changes in the alveolar epithelium in response to mycobacterial infection.

Electron microscopic examination of the lung of mice infected with a virulent strain of M. bovis (Ravenel) revealed marked alterations in the alveolar epithelial cells, particularly Type 2 cells (granular pneumocytes), in addition to the development of interstitial and intra-alveolar granuloma. Unlike the feature in uninfected mice, more than one Type 2 epithelial cells were often found adjacent to one another within a single alveolus. Some of these cells showed mitotic figures. Their characteristic lamellar inclusions were morphologically altered.