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.     

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.     

Glyphosate and nickel differently affect photosynthesis and ethylene in glyphosate-resistant soybean plants infected by Phakopsora pachyrhizi

Nickel (Ni) and glyphosate (Gl) are able to reduce the symptoms of Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, in soybean. However, their combined effects on the energy balance and ethylene metabolism of soybean plants infected with this fungus has not been elucidated. Therefore, the effects of Ni, Gl, and the combination of Ni + Gl on ASR development, photosynthetic capacity, sugar concentrations, and ethylene concentrations in plants of a Gl-resistant cultivar, uninfected or infected with P. pachyrhizi, were investigated. Inoculated plants supplied with Ni had the highest foliar Ni concentration in all the treatments. Gl had a negative effect on the foliar Ni concentration in Ni-sprayed plants. The ASR severity was reduced in plants sprayed with Ni and Gl. Carotenoid and chlorophyll concentrations were higher in inoculated Ni, Gl, and Ni + Gl plants than in control plants. Based on the chlorophyll a fluorescence parameters, the photosynthetic apparatus of the control inoculated plants was damaged, and the least amount of energy was directed to the photochemistry process in these plants. The reduced capacity of the photosynthetic mechanism to capture light and use the energy absorbed by photosystem II in inoculated plants was reflected in their reduced capacity to process CO₂, as indicated by the high internal CO₂ concentrations and low rates of net carbon assimilation. The low sugar concentrations in inoculated plants from the control treatment were linked to their reduced photosynthetic capacity due to the high ASR severity. In uninfected plants, the ethylene concentration was not affected by Ni or Gl, while the ethylene concentration decreased in inoculated plants; this decrease was more pronounced in plants from the control treatment than in treated inoculated plants. In conclusion, this study sheds light on the role played by both Ni and Gl in ASR control from a physiological perspective. Soybean plants exposed to Ni and Gl were able to maintain high ethylene concentrations and photosynthetic capacity during the P. pachyrhizi infection process; as a result, these plants consumed less of their reserves than inoculated plants not treated with Ni or Gl.     

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.     

The Lesion Simulating Disease (LSD) gene family as a variable in soybean response to Phakopsora pachyrhizi infection and dehydration

The Lesion Simulating Disease (LSD) genes encode a family of zinc finger proteins that are reported to play an important role in the hypersensitive response and programmed cell death (PCD) that are caused by biotic and abiotic stresses. In the present study, 117 putative LSD family members were identified in Viridiplantae. Genes with one, two, or three conserved LSD domains were identified. Proteins with three LSD domains were highly represented in the species analyzed and were present in basal organisms. Proteins with two LSD domains were identified only in the Embryophyte clade, and proteins possessing one LSD domain were highly represented in grass species. Expression analyses of Glycine max LSD (GmLSD) genes were performed by real-time quantitative polymerase chain reaction. The results indicated that GmLSD genes are not ubiquitously expressed in soybean organs and that their expression patterns are instead organ-dependent. The expression of the majority of GmLSD genes is modulated in soybean during Phakopsora pachyrhizi infection. In addition, the expression of some GmLSD genes is modulated in plants under dehydration stress. These results suggest the involvement of GmLSD genes in the response of soybean to both biotic and abiotic stresses.     

Protective gene expression changes elicited by an inherited defect in photoreceptor structure

Inherited defects in retinal photoreceptor structure impair visual transduction, disrupt relationship with the retinal pigment epithelium (RPE), and compromise cell viability. A variety of progressive retinal degenerative diseases can result, and knowledge of disease etiology remains incomplete. To investigate pathogenic mechanisms in such instances, we have characterized rod photoreceptor and retinal gene expression changes in response to a defined insult to photoreceptor structure, using the retinal degeneration slow (rds) mouse model. Global gene expression profiling was performed on flow-sorted rds and wild-type rod photoreceptors immediately prior and subsequent to times at which OSs are normally elaborated. Dysregulated genes were identified via microarray hybridization, and selected candidates were validated using quantitative PCR analyses. Both the array and qPCR data revealed that gene expression changes were generally modest and dispersed amongst a variety of known functional networks. Although genes showing major (>5-fold) differential expression were identified in a few instances, nearly all displayed transient temporal profiles, returning to WT levels by postnatal day (P) 21. These observations suggest that major defects in photoreceptor cell structure may induce early homeostatic responses, which function in a protective manner to promote cell viability. We identified a single key gene, Egr1, that was dysregulated in a sustained fashion in rds rod photoreceptors and retina. Egr1 upregulation was associated with microglial activation and migration into the outer retina at times subsequent to the major peak of photoreceptor cell death. Interestingly, this response was accompanied by neurotrophic factor upregulation. We hypothesize that activation of Egr1 and neurotrophic factors may represent a protective immune mechanism which contributes to the characteristically slow retinal degeneration of the rds mouse model.     

Nucleotide polymorphism in the 5.8S nrDNA gene and internal transcribed spacers in Phakopsora pachyrhizi viewed from structural models

The assessment of nucleotide polymorphisms in environmental samples of obligate pathogens requires DNA amplification through the polymerase chain reaction (PCR) and bacterial cloning of PCR products prior to sequencing. The drawback of this strategy is that it can give rise to false polymorphisms owing to DNA polymerase misincorporation during PCR or bacterial cloning. We investigated patterns of nucleotide polymorphism in the internal transcribed spacer (ITS) region for Phakopsora pachyrhizi, an obligate biotrophic fungus that causes the Asian soybean rust. Field-collected samples of P. pachyrhizi were obtained from all major soybean production areas worldwide, including Brazil and the United States. Bacterially-cloned, PCR products were obtained using a high fidelity DNA polymerase. A total of 370 ITS sequences that were subjected to an array of complementary sequence analyses, which included analyses of secondary structure stability, the pattern of nucleotide polymorphisms, GC content, and the presence of conserved motifs. The sequences exhibited features of functional rRNAs. Overall, polymorphisms took place within less conserved motives, such as loops and bulges; alternatively, they gave rise to non-canonical G-U pairs within conserved regions of double stranded helices. We discuss the usefulness of structural analyses to filter out putative 'suspicious' bacterially cloned ITS sequences, thus keeping artificially-induced sequence variation to a minimum.     

Elevated CO2 atmosphere enhances production of defense-related flavonoids in soybean elicited by NO and a fungal elicitor

Increased atmospheric pollutants including carbon dioxide (CO₂) and nitric oxide (NO) have a large impact on vegetation, with detrimental or beneficial influences on plant growth and metabolism. Here, we evaluated the effect of an elevated CO₂ atmosphere on the production of soybean defensive secondary chemicals induced by NO and a fungal elicitor. We hypothesized that an excess of carbon may alter the production of specific flavonoids that were previously shown to be induced by NO in soybean cotyledons. Pots containing soybean seeds (Glycine max [L.] Merr.) were submitted to 380 and 760 μmol mol^?1 of atmospheric CO₂ in open-top chambers. After nine days, plantlets grown under these conditions were assessed for biochemical and physiological parameters. Defense-related flavonoids were evaluated in detached cotyledon diffusates elicited with two different NO donors and with the β-glucan elicitor from Phytophthora sojae. A CO₂-enriched atmosphere stimulated initial growth, photosynthetic assimilation, and an altered C/N ratio in soybean plantlets resulting in allocation of precursors into different branches of the phenylpropanoid pathway in the cotyledons. Under elevated CO₂, the biotic elicitor caused accumulation of phytoalexins (glyceollins) as the natural end products of the phenylpropanoid pathway. In contrast, elevated CO₂ combined with NO resulted in an increase of intermediates and diverted end products (daidzein—127%, coumestrol—93%, genistein—93%, luteolin—89% and apigenin—238%) with a concomitant increase of 1.5–3.0 times in the activity of enzymes related to their biosynthetic routes. These observations point to changes in the pool of defense-related flavonoids that are related to increased carbon availability in soybeans. This may alter the responsiveness of soybean plants to pathogens when they are grown in CO₂ atmospheric concentrations close to those predicted for the upcoming several decades.     

Preventive and protective effects of silymarin on doxorubicin-induced testicular damages correlate with changes in c-myc gene expression

This study aimed to investigate the preventive and protective effects of silymarin (SMN) on doxorubicin (DOX)-induced damages in the testis. Wistar rats were divided into six groups (n=8), including: control (C), DOX-treated (DOX, 15mg/kg, i.p.), DOX- and SMN-treated and SMN-treated animals (SMN, 50mg/kg, orally). Those groups, which received either compounds, were sub-grouped based on the preventive (PVT), protective (PTT) and/or therapeutic regimens (TPT) of SMN administration. The antioxidant status analyses, hormonal assay, and histopathological examinations in the testis were conducted. The expression of c-myc at mRNA level also was analyzed. SMN in preventive and protective forms significantly (p<0.05) improved the DOX-induced weight loss and lowered the alkaline phosphatase level. Pretreatment and co-treatment with SMN attenuated the DOX-induced carbonyl stress. The DOX-induced histopathological damages including negative TDI and IR were significantly (p<0.05) improved with SMN pretreatment and co-administration. SMN in preventive and protective forms prevented from DOX-induced DNA fragmentation in the testis. SMN ameliorated the DOX-reduced serum level of sexual hormones including testosterone, inhibin B, LH and FSH in PVT and PTT groups. The c-myc expression at mRNA level was completely and relatively down regulated in the testis of animals that received SMN as pretreatment and concurrent administration, respectively. Our data suggests that the DOX-induced biochemical and histopathological alterations could be prevented and/or protected by SMN. Moreover, the SMN protective and preventive effects attribute to its capacity in the reduction of DOX-induced carbonyl stress and DNA damage, which may be mediated by c-myc expression.     

Effects of temperature during soybean seed development on defense-related gene expression and fungal pathogen accumulation

Soybean [Glycine max (L.) Merr] plants were exposed to three temperature regimens during seed development to investigate the effect of temperature on the expression of eight defense-related genes and the accumulation of two fungal pathogens in inoculated seeds. In seeds prior to inoculation, either a day/night warm (34/26°C) or a cool temperature (22/18°C) relative to normal (26/22°C) resulted in altered patterns of gene expression including substantially lower expression of PR1, PR3 and PR10. After seed inoculation with Cercospora kikuchii, pathogen accumulation was lowest in seeds produced at 22/18°C in which of all defense genes, MMP2 was uniquely most highly induced. For seeds inoculated with Diaporthe phaseolorum, pathogen accumulation was lowest in seeds produced at 34/26°C in which of all defense genes, PR10 was uniquely most highly induced. Our detached seed assays clearly demonstrated that the temperature regimens we applied during seed development produced significant changes in seed defense-related gene expression both pre- and post inoculation and our findings support the hypothesis that global climate change may alter plant–pathogen interactions and thereby potentially crop productivity.     

Cadmium-induced changes in growth and cell cycle gene expression in suspension-culture cells of soybean

The toxic effects of cadmium on growth and development of living organisms are well documented. However, the molecular mechanisms responsible for the inhibition of plant growth by cadmium are still not completely understood. We determined the effects of cadmium concentrations in the range of 1–11 μM on the growth of Glycine max L. cv. Navico suspension-culture cells, as well as on the expression of two cell cycle genes: cyclin B1 and cyclin-dependent type A kinase (CDK-A). There was no detectable decrease in cell viability at any tested Cd²⁺ concentrations. The lower concentrations of Cd²⁺ (1–4 μM) stimulated cell culture growth; however, this did not correspond with increased expression of cell cycle genes. The inhibition of cell growth was observed at concentration of Cd²⁺ higher than 6 μM. Interestingly, it correlated well with the decreased cyclin B1 mRNA levels, but had no significant effect on the levels of CDK-A mRNA.     

In plants, expression breadth and expression level distinctly and non-linearly correlate with gene structure

Background  

Compactness of highly/broadly expressed genes in human has been explained as selection for efficiency, regional mutation biases or genomic design. However, highly expressed genes in flowering plants were shown to be less compact than lowly expressed ones. On the other hand, opposite facts have also been documented that pollen-expressed Arabidopsis genes tend to contain shorter introns and highly expressed moss genes are compact. This issue is important because it provides a chance to compare the selectionism and the neutralism views about genome evolution. Furthermore, this issue also helps to understand the fates of introns, from the angle of gene expression.     

In plants, expression breadth and expression level distinctly and non-linearly correlate with gene structure

Background

Staphylococcus aureus is a major human pathogen responsible for a variety of nosocomial and community-acquired infections. Recent reports show that the prevalence of Methicillin-Resistant S. aureus (MRSA) infections in cystic fibrosis (CF) patients is increasing. In 2006 in Marseille, France, we have detected an atypical MRSA strain with a specific antibiotic susceptibility profile and a unique growth phenotype. Because of the clinical importance of the spread of such strain among CF patients we decided to sequence the genome of one representative isolate (strain CF-Marseille) to compare this to the published genome sequences. We also conducted a retrospective epidemiological analysis on all S. aureus isolated from 2002 to 2007 in CF patients from our institution.

Results

CF-Marseille is multidrug resistant, has a hetero-Glycopeptide-Intermediate resistance S. aureus phenotype, grows on Cepacia agar with intense orange pigmentation and has a thickened cell wall. Phylogenetic analyses using Complete Genome Hybridization and Multi Locus VNTR Assay showed that CF-Marseille was closely related to strain Mu50, representing vancomycin-resistant S. aureus. Analysis of CF-Marseille shows a similar core genome to that of previously sequenced MRSA strains but with a different genomic organization due to the presence of specific mobile genetic elements i.e. a new SCCmec type IV mosaic cassette that has integrated the pUB110 plasmid, and a new phage closely related to phiETA3. Moreover this phage could be seen by electron microscopy when mobilized with several antibiotics commonly used in CF patients including, tobramycin, ciprofloxacin, cotrimoxazole, or imipenem. Phylogenetic analysis of phenotypically similar h-GISA in our study also suggests that CF patients are colonized by polyclonal populations of MRSA that represents an incredible reservoir for lateral gene transfer.

Conclusion

In conclusion, we demonstrated the emergence and spreading of a new isolate of MRSA in CF patients in Marseille, France, that has probably been selected in the airways by antibiotic pressure. Antibiotic-mediated phage induction may result in high-frequency transfer and the unintended consequence of promoting the spread of virulence and/or antibiotic resistance determinants. The emergence of well-adapted MRSA is worrying in such population chronically colonized and receiving many antibiotics and represents a model for emergence of uncontrollable super bugs in a specific niche.

Reviewers

This article was reviewed by Eric Bapteste, Pierre Pontarotti, and Igor Zhulin. For the full reviews, please go to the Reviewers' comments section.     

Changes in cell shape correlate with collagenase gene expression in rabbit synovial fibroblasts

Induction of the neutral proteinase, collagenase, is a marker for a specific switch in gene expression observed in rabbit synovial fibroblasts. A variety of agents, including 12-O-tetradecanoylphorbol-13-acetate, cytochalasins B and D, trypsin, chymotrypsin, poly(2-hydroxyethylmethacrylate), and trifluoperazine induced this change in gene expression. Induction of collagenase by these agents was always correlated with a marked alteration in cell morphology, although the cells remained adherent to the culture dishes. The amount of collagenase induced was positively correlated with the degree of shape change produced by a given concentration and, to some extent, with the duration of treatment. Altered cell morphology was required only during the first few hours of treatment with inducing agents; after this time collagenase synthesis continued for up to 6 d even when agents were removed and normal flattened cell morphology was regained. All agents that altered cell morphology also produced a characteristic switch in protein secretion phenotype, characterized by the induction of procollagenase (Mr 53,000 and 57,000) and a neutral metalloproteinase (Mr 51,000), which accounted for approximately 25% and 15% of the protein secreted, respectively. Secretion of another neutral proteinase, plasminogen activator, did not correlate with increased collagenase secretion. In contrast, synthesis and secretion of a number of other polypeptides, including the extracellular matrix proteins, collagen and fibronectin, were concomitantly decreased. That changes in cell shape correlated with a program of gene expression manifested by both degradation and synthesis of extracellular macromolecules may have broad implications in development, repair, and pathologic conditions.