Epigenetic insight into regulatory role of chromatin covalent modifications in lifecycle and virulence of Phytophthora.

The Oomycota phylum includes fungi-like filamentous microorganisms classified as plant pathogens. The most destructive genus within oomycetes is Phytophthora, which causes diseases in plants of economic importance in agriculture, forestry and ornamental. Phytophthora species are widespread worldwide and some of them enable adaptation to different hosts and environmental changes. The development of sexual and asexual reproductive structures and the secretion of proteins to control plant immunity are critical for the adaptative lifestyle. However, molecular mechanisms underlying the adaptation of Phytophthora to different hosts and environmental changes are poorly understood. In the last decade, the role of epigenetics has gained attention, and important evidence has demonstrated the potential role of chromatin covalent modifications, such as DNA methylation and histone acetylation/methylation, in the regulation of gene expression during Phytophthora development and plant infection. Here, we review for the first time the evidence of the potential role of chromatin covalent modifications in the lifecycle of the phytopathogenic genus Phytophthora, including virulence, and host and environment adaptation processes.

Cell death in Ustilago maydis: comparison with other fungi and the effect of metformin and curcumin on its chronological lifespan.

Ustilago maydis is a Basidiomycota fungus, in which very little is known about its mechanisms of cell survival and death. To date, only the role of metacaspase1, acetate and hydrogen peroxide as inducers of cell death has been investigated. In the present work, we analyzed the lifespan of U. maydis compared with other species like Sporisorium reilianum, Saccharomyces cerevisiae and Yarrowia lipolytica, and we observed that U. maydis has a minor lifespan. We probe the addition of low concentrations metformin and curcumin to the culture media, and we observed that both prolonged the lifespan of U. maydis, a result observed for the first time in a phytopathogen fungus. However, higher concentrations of curcumin were toxic for the cells, and interestingly induced the yeast-to-mycelium dimorphic transition. The positive effect of metformin and curcumin appears to be related to an inhibition of the mechanistic Target of Rapamycin (mTOR) pathway, increase expression of autophagy genes and reducing of reactive oxygen species. These data indicate that U. maydis may be a eukaryotic model organism to elucidate the molecular mechanism underlying apoptotic and necrosis pathways, and the lifespan increase caused by metformin and curcumin.

Autophagosomes accumulation in the vacuoles of the fungus Ustilago maydis and the role of proteases in their digestion.

In the present study we determined whether Ustilago maydis accumulates autophagosomes within vacuoles when the cells are exposed to nutritional stress conditions. We investigated whether proteinase B and proteinase A are involved in their degradation. To this effect, wild type and Δpep4 mutant were incubated in minimal medium lacking a carbon source. It was observed that after incubation in nutrient-deficient media, spherical bodies appeared within the Δpep4 mutant strains vacuoles. In addition, autophagosomes were accumulated in U. maydis WT cells incubated in the presence of the serine protease inhibitor PMSF and accumulation of large autophagosomes and electrodense structures in the Δpep4 mutant cell vacuoles took place. These results demonstrate that the homologues of both, the proteinase B and the protease A, are involved in the autophagosomes degradation process in U. maydis.

Non-host Plant Resistance against Phytophthora capsici Is Mediated in Part by Members of the I2 R Gene Family in Nicotiana spp.

The identification of host genes associated with resistance to Phytophthora capsici is crucial to developing strategies of control against this oomycete pathogen. Since there are few sources of resistance to P. capsici in crop plants, non-host plants represent a promising source of resistance genes as well as excellent models to study P. capsici - plant interactions. We have previously shown that non-host resistance to P. capsici in Nicotiana spp. is mediated by the recognition of a specific P. capsici effector protein, PcAvr3a1 in a manner that suggests the involvement of a cognate disease resistance (R) genes. Here, we have used virus-induced gene silencing (VIGS) and transgenic tobacco plants expressing dsRNA in Nicotiana spp. to identify candidate R genes that mediate non-host resistance to P. capsici. Silencing of members of the I2 multigene family in the partially resistant plant N. edwardsonii and in the resistant N. tabacum resulted in compromised resistance to P. capsici. VIGS of two other components required for R gene-mediated resistance, EDS1 and SGT1, also enhanced susceptibility to P. capsici in N. edwardsonii, as well as in the susceptible plants N. benthamiana and N. clevelandii. The silencing of I2 family members in N. tabacum also compromised the recognition of PcAvr3a1. These results indicate that in this case, non-host resistance is mediated by the same components normally associated with race-specific resistance.

Analysis of the tomato leaf transcriptome during successive hemibiotrophic stages of a compatible interaction with the oomycete pathogen Phytophthora infestans.

The infection of plants by hemibiotrophic pathogens involves a complex and highly regulated transition from an initial biotrophic, asymptomatic stage to a later necrotrophic state, characterized by cell death. Little is known about how this transition is regulated, and there are conflicting views regarding the significance of the plant hormones jasmonic acid (JA) and salicylic acid (SA) in the different phases of infection. To provide a broad view of the hemibiotrophic infection process from the plant perspective, we surveyed the transcriptome of tomato (Solanum lycopersicum) during a compatible interaction with the hemibiotrophic oomycete Phytophthora infestans during three infection stages: biotrophic, the transition from biotrophy to necrotrophy, and the necrotrophic phase. Nearly 10 000 genes corresponding to proteins in approximately 400 biochemical pathways showed differential transcript abundance during the three infection stages, revealing a major reorganization of plant metabolism, including major changes in source-sink relations, as well as secondary metabolites. In addition, more than 100 putative resistance genes and pattern recognition receptor genes were induced, and both JA and SA levels and associated signalling pathways showed dynamic changes during the infection time course. The biotrophic phase was characterized by the induction of many defence systems, which were either insufficient, evaded or suppressed by the pathogen.

Transcriptional dynamics of Phytophthora infestans during sequential stages of hemibiotrophic infection of tomato.

Hemibiotrophic plant pathogens, such as the oomycete Phytophthora infestans, employ a biphasic infection strategy, initially behaving as biotrophs, where minimal symptoms are exhibited by the plant, and subsequently as necrotrophs, feeding on dead plant tissue. The regulation of this transition and the breadth of molecular mechanisms that modulate plant defences are not well understood, although effector proteins secreted by the pathogen are thought to play a key role. We examined the transcriptional dynamics of P. infestans in a compatible interaction with its host tomato (Solanum lycopersicum) at three infection stages: biotrophy; the transition from biotrophy to necrotrophy; and necrotrophy. The expression data suggest a tight temporal regulation of many pathways associated with the suppression of plant defence mechanisms and pathogenicity, including the induction of putative cytoplasmic and apoplastic effectors. Twelve of these were experimentally evaluated to determine their ability to suppress necrosis caused by the P. infestans necrosis-inducing protein PiNPP1.1 in Nicotiana benthamiana. Four effectors suppressed necrosis, suggesting that they might prolong the biotrophic phase. This study suggests that a complex regulation of effector expression modulates the outcome of the interaction.

Recognition of an Avr3a homologue plays a major role in mediating nonhost resistance to Phytophthora capsici in Nicotiana species.

Nonhost resistance is a commonly occurring phenomenon wherein all accessions or cultivars of a plant species are resistant to all strains of a pathogen species and is likely the manifestation of multiple molecular mechanisms. Phytophthora capsici is a soil-borne oomycete that causes Phytophthora blight disease in many solanaceous and cucurbitaceous plants worldwide. Interest in P. capsici has increased considerably with the sequencing of its genome and its increasing occurrence in multiple crops. However, molecular interactions between P. capsici and both its hosts and its nonhosts are poorly defined. We show here that tobacco (Nicotiana tabacum) acts like a nonhost for P. capsici and responds to P. capsici infection with a hypersensitive response (HR). Furthermore, we have found that a P. capsici Avr3a-like gene (PcAvr3a1) encoding a putative RXLR effector protein produces a HR upon transient expression in tobacco and several other Nicotiana species. This HR response correlated with resistance in 19 of 23 Nicotiana species and accessions tested, and knock-down of PcAvr3a1 expression by host-induced gene silencing allowed infection of resistant tobacco. Our results suggest that many Nicotiana species have the capacity to recognize PcAvr3a1 via the products of endogenous disease resistance (R) genes and that this R gene-mediated response is a major component of nonhost resistance to P. capsici.

Distinctive expansion of potential virulence genes in the genome of the oomycete fish pathogen Saprolegnia parasitica.

Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinkler's, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.

Identification of an ARGONAUTE for antiviral RNA silencing in Nicotiana benthamiana.

ARGONAUTE proteins (AGOs) are known to be key components of the RNA silencing mechanism in eukaryotes that, among other functions, serves to protect against viral invaders. Higher plants encode at least 10 individual AGOs yet the role played by many in RNA silencing-related antiviral defense is largely unknown, except for reports that AGO1, AGO2, and AGO7 play an antiviral role in Arabidopsis (Arabidopsis thaliana). In the plant virus model host Nicotiana benthamiana, Tomato bushy stunt virus (TBSV) P19 suppressor mutants are very susceptible to RNA silencing. Here, we report that a N. benthamiana AGO (NbAGO) with similarity to Arabidopsis AGO2, is involved in antiviral defense against TBSV. The activity of this NbAGO2 is shown to be directly associated with anti-TBSV RNA silencing, while its inactivation does not influence silencing of transiently expressed transgenes. Thus, the role of NbAGO2 might be primarily for antiviral defense.

The Palomero genome suggests metal effects on domestication.

Maize domestication (Zea mays ssp. mays L.) resulted in a wide diversity of native landraces that represent an invaluable source of genetic information for exploring natural variation and genome evolution. We sequenced de novo the approximately 2-gigabase genome of the Mexican landrace Palomero Toluqueño (Palomero) and compared its features to those of the modern inbred line B73. We revealed differences concordant with its ancient origin and identified chromosomal regions of low nucleotide variability that contain domestication genes involved in heavy-metal detoxification. Our results indicate that environmental changes were important selective forces acting on maize domestication.

Deep sampling of the Palomero maize transcriptome by a high throughput strategy of pyrosequencing.

BACKGROUND: In-depth sequencing analysis has not been able to determine the overall complexity of transcriptional activity of a plant organ or tissue sample. In some cases, deep parallel sequencing of Expressed Sequence Tags (ESTs), although not yet optimized for the sequencing of cDNAs, has represented an efficient procedure for validating gene prediction and estimating overall gene coverage. This approach could be very valuable for complex plant genomes. In addition, little emphasis has been given to efforts aiming at an estimation of the overall transcriptional universe found in a multicellular organism at a specific developmental stage. RESULTS: To explore, in depth, the transcriptional diversity in an ancient maize landrace, we developed a protocol to optimize the sequencing of cDNAs and performed 4 consecutive GS20-454 pyrosequencing runs of a cDNA library obtained from 2 week-old Palomero Toluqueño maize plants. The protocol reported here allowed obtaining over 90% of informative sequences. These GS20-454 runs generated over 1.5 Million reads, representing the largest amount of sequences reported from a single plant cDNA library. A collection of 367,391 quality-filtered reads (30.09 Mb) from a single run was sufficient to identify transcripts corresponding to 34% of public maize ESTs databases; total sequences generated after 4 filtered runs increased this coverage to 50%. Comparisons of all 1.5 Million reads to the Maize Assembled Genomic Islands (MAGIs) provided evidence for the transcriptional activity of 11% of MAGIs. We estimate that 5.67% (86,069 sequences) do not align with public ESTs or annotated genes, potentially representing new maize transcripts. Following the assembly of 74.4% of the reads in 65,493 contigs, real-time PCR of selected genes confirmed a predicted correlation between the abundance of GS20-454 sequences and corresponding levels of gene expression. CONCLUSION: A protocol was developed that significantly increases the number, length and quality of cDNA reads using massive 454 parallel sequencing. We show that recurrent 454 pyrosequencing of a single cDNA sample is necessary to attain a thorough representation of the transcriptional universe present in maize, that can also be used to estimate transcript abundance of specific genes. This data suggests that the molecular and functional diversity contained in the vast native landraces remains to be explored, and that large-scale transcriptional sequencing of a presumed ancestor of the modern maize varieties represents a valuable approach to characterize the functional diversity of maize for future agricultural and evolutionary studies.

Arabidopsis thaliana is a host of the legume nanovirus Faba bean necrotic yellows virus.

We report infection of Arabidopsis thaliana with the legume nanovirus Faba bean necrotic yellows virus (FBNYV) by its insect vector Aphis craccivora. Symptoms of FBNYV infection on A. thaliana include stunting and reduced apical dominance, and are rather mild, compared to the severe necrosis and early plant death induced by the virus in the natural host Vicia faba. An inoculation access period of 6h is sufficient to transmit FBNYV to A. thaliana. FBNYV is readily transmitted back from A. thaliana to V. faba, where it induces the characteristic severe disease symptoms. Hence, passage through A. thaliana does not affect FBNYV pathogenicity. FBNYV accumulates to the highest levels in roots and stems, compared to cauline and rosette leaves. In cauline leaves, the kinetics of virus accumulation correlates with the amount of master Rep protein accumulation.

A functional histidine-tagged replication initiator protein: implications for the study of single-stranded DNA virus replication in planta.

Replication initiation of nanoviruses, plant viruses with a multipartite circular single-stranded DNA genome, is triggered by the master Rep (M-Rep) protein. To enable the study of interactions between M-Rep and viral or host factors involved in replication, we designed oligohistidine-tagged variants of the nanovirus Faba bean necrotic yellows virus (FBNYV) M-Rep protein that allow affinity purification of enzymatically active M-Rep from plant tissue. The tagged M-Rep protein was able to initiate replication of its cognate and other FBNYV DNAs in Nicotiana benthamiana leaf disks and plants. The replicon encoding the tagged M-Rep protein multiplied and moved systemically in FBNYV-infected Vicia faba plants and was transmitted by the aphid vector of the virus. Using the tagged M-Rep protein, we demonstrated the in planta interaction between wild-type M-Rep and its tagged counterpart. Such a tagged and fully functional replication initiator protein will have bearings on the isolation of protein complexes from plants.