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.     

Prediction of the in planta Phakopsora pachyrhizi secretome and potential effector families

Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, can cause losses greater than 80%. Despite its economic importance, there is no soybean cultivar with durable ASR resistance. In addition, the P. pachyrhizi genome is not yet available. However, the availability of other rust genomes, as well as the development of sample enrichment strategies and bioinformatics tools, has improved our knowledge of the ASR secretome and its potential effectors. In this context, we used a combination of laser capture microdissection (LCM), RNAseq and a bioinformatics pipeline to identify a total of 36 350 P. pachyrhizi contigs expressed in planta and a predicted secretome of 851 proteins. Some of the predicted secreted proteins had characteristics of candidate effectors: small size, cysteine rich, do not contain PFAM domains (except those associated with pathogenicity) and strongly expressed in planta. A comparative analysis of the predicted secreted proteins present in Pucciniales species identified new members of soybean rust and new Pucciniales‐ or P. pachyrhizi‐specific families (tribes). Members of some families were strongly up‐regulated during early infection, starting with initial infection through haustorium formation. Effector candidates selected from two of these families were able to suppress immunity in transient assays, and were localized in the plant cytoplasm and nuclei. These experiments support our bioinformatics predictions and show that these families contain members that have functions consistent with P. pachyrhizi effectors.     

Expression patterns in soybean resistant to Phakopsora pachyrhizi reveal the importance of peroxidases and lipoxygenases

Soybean rust caused by Phakopsora pachyrhizi Sydow is a devastating foliar disease that has spread to most soybean growing regions throughout the world, including the USA. Four independent rust resistance genes, Rpp1–Rpp4, have been identified in soybean that recognize specific isolates of P. pachyrhizi. A suppressive subtraction hybridization (SSH) complementary DNA (cDNA) library was constructed from the soybean accession PI200492, which contains Rpp1, after inoculation with two different isolates of P. pachyrhizi that result in susceptible or immune reactions. Both forward and reverse SSH were performed using cDNA from messenger RNA pooled from 1, 6, 12, 24, and 48 h post-inoculation. A total of 1,728 SSH clones were sequenced and compared to sequences in GenBank for similarity. Microarray analyses were conducted on a custom 7883 soybean-cDNA clone array encompassing all of the soybean-rust SSH clones and expressed sequence tags from four other soybean cDNA libraries. Results of the microarray revealed 558 cDNA clones differentially expressed in the immune reaction. The majority of the upregulated cDNA clones fell into the functional category of defense. In particular, cDNA clones with similarity to peroxidases and lipoxygenases were prevalent. Downregulated cDNA clones included those with similarity to cell-wall-associated protein, such as extensins, proline-rich proteins, and xyloglucan endotransglycosylases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Leaf gas exchange and chlorophyll a fluorescence in soybean leaves infected by Phakopsora pachyrhizi

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is one of the most important foliar diseases affecting soybean production worldwide. This study aimed to investigate the photosynthetic performance (leaf gas exchange, chlorophyll (Chl) a fluorescence images and photosynthetic pigment pools) of soybean plants sprayed with Acibenzolar‐S‐Methyl (ASM) and the fungicide epoxiconazole + pyraclostrobin (Epo+Pyr) and further inoculated with P. pachyrhizi. The ASR symptoms progressed much faster on the leaves of plants from the control treatment (water spray) in comparison with the ASM and Epo+Pyr treatments. In general, the values for the leaf gas exchange parameters net carbon assimilation rate (A), stomatal conductance to water vapour (g_s), internal CO₂ concentration (C_i) and transpiration rate (E) increased for the infected plants sprayed with ASM or Epo+Pyr in comparison with plants from the control treatment. The values for the initial fluorescence (F_o), maximal fluorescence (F_m), maximal photosystem II quantum efficiency (F_v/F_m), effective photosystem II quantum yield (Y(II)) and quantum yield of regulated energy dissipation (Y(NPQ)) were consistently higher for the ASM and Epo+Pyr treatments in comparison with the control treatment at advanced stages of fungal infection. By contrast, the values for quantum yield of non‐regulated energy dissipation (Y(NO) were significantly lower for the ASM and Epo+Pyr treatments. The concentrations of total Chl a+b and carotenoids significantly increased for infected plants sprayed with ASM and Epo+Pyr in comparison with plants from the control treatment. The results of this study demonstrated that the spray of soybean plants with either ASM or Epo+Pyr contributed to reduce the negative effect of ASR on the photosynthesis of soybean plants.     

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 .     

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.     

The Infection of Soybean Leaves by Phakopsora pachyrhizi during Conditions of Discontinuous Wetness

The ability of Phakopsora pachyrhizi to cause infection under conditions of discontinuous wetness was investigated. In in vitro experiments, droplets of a uredospore suspension were deposited onto the surface of polystyrene. After an initial wetting period of either 1, 2 or 4 h, the drops were dried for different time intervals and then the wetness was restored for 11, 10 or 8 h. Germination and appressorium formation were evaluated. In in vivo experiments, soybean plants were inoculated with a uredospore suspension. Leaf wetness was interrupted for 1, 3 or 6 h after initial wetting periods of 1, 2 or 4 h. Then, the wetting was re‐established for 11, 10 or 8 h, respectively. Rust severity was evaluated 14 days after inoculation. The germination of the spores and the formation of the appressoria on the soybean leaves after different periods of wetness were also quantified in vivo by scanning electron microscopy. P. pachyrhizi showed a high infective capacity during short periods of time. An interruption of wetness after 1 h caused average reductions in germination from 56 to 75% and in appressorium formation from 84 to 96%. Rust severity was lower in all of the in vivo treatments with discontinuous wetness when compared to the control plants. Rust severity was zero when the interruption of wetness occurred 4 h after the initial wetting. Wetting interruptions after 1 and 2 h reduced the average rust severity by 83 and 77%, respectively. The germination of the uredospores on the soybean leaves occurred after 2 h of wetness, with a maximum germination appearing after 4 h of wetness. Wetness interruption affected mainly the spores that had initiated the germination.     

Roles of small RNAs in soybean defense against Phytophthora sojae infection

The genus Phytophthora consists of many notorious pathogens of crops and forestry trees. At present, battling Phytophthora diseases is challenging due to a lack of understanding of their pathogenesis. We investigated the role of small RNAs in regulating soybean defense in response to infection by Phytophthora sojae, the second most destructive pathogen of soybean. Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are universal regulators that repress target gene expression in eukaryotes. We identified known and novel small RNAs that differentially accumulated during P. sojae infection in soybean roots. Among them, miR393 and miR166 were induced by heat‐inactivated P. sojae hyphae, indicating that they may be involved in soybean basal defense. Indeed, knocking down the level of mature miR393 led to enhanced susceptibility of soybean to P. sojae; furthermore, the expression of isoflavonoid biosynthetic genes was drastically reduced in miR393 knockdown roots. These data suggest that miR393 promotes soybean defense against P. sojae. In addition to miRNAs, P. sojae infection also resulted in increased accumulation of phased siRNAs (phasiRNAs) that are predominantly generated from canonical resistance genes encoding nucleotide binding‐leucine rich repeat proteins and genes encoding pentatricopeptide repeat‐containing proteins. This work identifies specific miRNAs and phasiRNAs that regulate defense‐associated genes in soybean during Phytophthora infection.     

Proteomic analysis of rice seedlings infected by Sinorhizobium meliloti 1021

Rhizobial endophytes infect and colonize not only leguminous plants, but several non‐leguminous species as well. Using green fluorescent protein tagging technique, it has been shown that Rhizobia infect different varieties of rice species and migrate from plant roots to aerial tissues such as leaf sheaths and leaves. The interaction between them was found to promote the growth of rice. The growth promotion is the cumulative result of enhanced photosynthesis and stress resistance. In addition, indole‐3‐acetic acid also contributes to the promotion. Gel‐based comparative proteomic approaches were applied to analyze the protein profiles of three different tissues (root, leaf sheath and leaf) of Sinorhizobium meliloti 1021 inoculated rice in order to get an understanding about the molecular mechanism. Upon the inoculation of rhizobia, proteins involved in nine different functional categories were either up‐regulated or down‐regulated. Photosynthesis related proteins were up‐regulated only in leaf sheath and leaf, while the up‐regulated proteins in root were exclusively defense related. The results implied that there might have been an increase in the import and transport of proteins involved in light and dark reactions to the chloroplast as well as more efficient distribution of nutrients, hence enhanced photosynthesis. Although the initiation of defensive reactions mainly occurred in roots, some different defense mechanisms were also evoked in the aerial tissues.     

Defense Reactions in Host and Nonhost Plants Against the Soybean Rust Fungus (Phakopsora pachyrhizi Syd.)

Growth and development of two races of the soybean rust fungus (Phakopsora pachyrhizi Syd.) were compared on host and nonhost plants. Both groups had several lines of defense, each of which could stop a part of attacking uredospores. Germ tubes and appressoria were produced equally well on hosts and nonhosts. A reduced formation of penetration hyphae contributed to the resistance of nonhosts and resistant host genotypes. In the epidermal cells of wheat and barley leaves, lower frequencies of penetration hyphae coincided with the production of papillae-like structures which were not penetrated. The last line of defense of all nonhosts was localized in the epidermal cell where the growth of the penetration hyphae was arrested definitively. The colony development in these species was suppressed completely. In highly resistant host genotypes the last defense reaction occurred later as a hypersensitive cell collapse which interrupted the growth of the rust colony.     

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.     

Assembly and annotation of a draft genome sequence for Glycine latifolia,a perennial wild relative of soybean

Glycine latifolia (Benth.) Newell & Hymowitz (2n = 40), one of the 27 wild perennial relatives of soybean, possesses genetic diversity and agronomically favorable traits that are lacking in soybean. Here, we report the 939‐Mb draft genome assembly of G. latifolia (PI 559298) using exclusively linked‐reads sequenced from a single Chromium library. We organized scaffolds into 20 chromosome‐scale pseudomolecules utilizing two genetic maps and the Glycine max (L.) Merr. genome sequence. High copy numbers of putative 91‐bp centromere‐specific tandem repeats were observed in consecutive blocks within predicted pericentromeric regions on several pseudomolecules. No 92‐bp putative centromeric repeats, which are abundant in G. max, were detected in G. latifolia or Glycine tomentella. Annotation of the assembled genome and subsequent filtering yielded a high confidence gene set of 54 475 protein‐coding loci. In comparative analysis with five legume species, genes related to defense responses were significantly overrepresented in Glycine‐specific orthologous gene families. A total of 304 putative nucleotide‐binding site (NBS)‐leucine‐rich‐repeat (LRR) genes were identified in this genome assembly. Different from other legume species, we observed a scarcity of TIR‐NBS‐LRR genes in G. latifolia. The G. latifolia genome was also predicted to contain genes encoding 367 LRR‐receptor‐like kinases, a family of proteins involved in basal defense responses and responses to abiotic stress. The genome sequence and annotation of G. latifolia provides a valuable source of alternative alleles and novel genes to facilitate soybean improvement. This study also highlights the efficacy and cost‐effectiveness of the application of Chromium linked‐reads in diploid plant genome de novo assembly.     

Mycoparasitism of Phakopsora pachyrhizi,the soybean rust pathogen,by Simplicillium lanosoniveum

Rust of soybean, caused by Phakopsora pachyrhizi, was first reported in the southeastern US in 2004 where it quickly became established. Yield losses ranged from 35% to more than 80%. The mycophilic fungus Simplicillium lanosoniveum was previously shown to colonize rust pustules on soybean leaves and prevent germination of urediniospores. The number of pustules on soybean leaves also was significantly reduced when S. lanosoniveum colonized leaves. This study examined the antagonistic interactions between P. pachyrhizi and S. lanosoniveum using confocal, transmission (TEM) and scanning electron microscopy (SEM) in order to determine if S. lanosoniveum was mycoparasitic. Co-inoculated detached soybean leaves were examined using TEM and SEM to examine changes in urediniospores colonized by S. lanosoniveum. An isolate of S. lanosoniveum, previously shown to be an antagonist of P. pachyrhizi, was transformed with the green fluorescent protein (GFP) gene and used to document the infection process using confocal microscopy. S. lanosoniveum colonized pustules and coiled around urediniospores before infection. The mycoparasite penetrated urediniospores through germ pores within 24 h after inoculation, at which time organelles showed signs of degradation. By 2 days after inoculation, there was extensive colonization of urediniospores by hyphae of S. lanosoniveum. Hyphae of the mycoparasite erupted from the colonized urediniospores at 7 days after inoculation, and there was extensive sporulation on the surface of urediniospores. Over 90% of urediniospores were colonized within 5 days after inoculation with the mycoparasite. We showed in previous studies that S. lanosoniveum is an antagonist of P. pachyrhizi as well as an effective biocontrol agent, but we were unable to document its mode of action. We now present microscopic evidence that S. lanosoniveum is a mycoparasite.