Comparative genome analyses of four rice-infecting Rhizoctonia solani isolates reveal extensive enrichment of homogalacturonan modification genes.

BACKGROUND: Plant pathogenic isolates of Rhizoctonia solani anastomosis group 1-intraspecific group IA (AG1-IA) infect a wide range of crops causing diseases such as rice sheath blight (ShB). ShB has become a serious disease in rice production worldwide. Additional genome sequences of the rice-infecting R. solani isolates from different geographical regions will facilitate the identification of important pathogenicity-related genes in the fungus. RESULTS: Rice-infecting R. solani isolates B2 (USA), ADB (India), WGL (India), and YN-7 (China) were selected for whole-genome sequencing. Single-Molecule Real-Time (SMRT) and Illumina sequencing were used for de novo sequencing of the B2 genome. The genomes of the other three isolates were then sequenced with Illumina technology and assembled using the B2 genome as a reference. The four genomes ranged from 38.9 to 45.0 Mbp in size, contained 9715 to 11,505 protein-coding genes, and shared 5812 conserved orthogroups. The proportion of transposable elements (TEs) and average length of TE sequences in the B2 genome was nearly 3 times and 2 times greater, respectively, than those of ADB, WGL and YN-7. Although 818 to 888 putative secreted proteins were identified in the four isolates, only 30% of them were predicted to be small secreted proteins, which is a smaller proportion than what is usually found in the genomes of cereal necrotrophic fungi. Despite a lack of putative secondary metabolite biosynthesis gene clusters, the rice-infecting R. solani genomes were predicted to contain the most carbohydrate-active enzyme (CAZyme) genes among all 27 fungal genomes used in the comparative analysis. Specifically, extensive enrichment of pectin/homogalacturonan modification genes were found in all four rice-infecting R. solani genomes. CONCLUSION: Four R. solani genomes were sequenced, annotated, and compared to other fungal genomes to identify distinctive genomic features that may contribute to the pathogenicity of rice-infecting R. solani. Our analyses provided evidence that genomic conservation of R. solani genomes among neighboring AGs was more diversified than among AG1-IA isolates and the presence of numerous predicted pectin modification genes in the rice-infecting R. solani genomes that may contribute to the wide host range and virulence of this necrotrophic fungal pathogen.

Perturbation of maize phenylpropanoid metabolism by an AvrE family type III effector from Pantoea stewartii.

AvrE family type III effector proteins share the ability to suppress host defenses, induce disease-associated cell death, and promote bacterial growth. However, despite widespread contributions to numerous bacterial diseases in agriculturally important plants, the mode of action of these effectors remains largely unknown. WtsE is an AvrE family member required for the ability of Pantoea stewartii ssp. stewartii (Pnss) to proliferate efficiently and cause wilt and leaf blight symptoms in maize (Zea mays) plants. Notably, when WtsE is delivered by a heterologous system into the leaf cells of susceptible maize seedlings, it alone produces water-soaked disease symptoms reminiscent of those produced by Pnss. Thus, WtsE is a pathogenicity and virulence factor in maize, and an Escherichia coli heterologous delivery system can be used to study the activity of WtsE in isolation from other factors produced by Pnss. Transcriptional profiling of maize revealed the effects of WtsE, including induction of genes involved in secondary metabolism and suppression of genes involved in photosynthesis. Targeted metabolite quantification revealed that WtsE perturbs maize metabolism, including the induction of coumaroyl tyramine. The ability of mutant WtsE derivatives to elicit transcriptional and metabolic changes in susceptible maize seedlings correlated with their ability to promote disease. Furthermore, chemical inhibitors that block metabolic flux into the phenylpropanoid pathways targeted by WtsE also disrupted the pathogenicity and virulence activity of WtsE. While numerous metabolites produced downstream of the shikimate pathway are known to promote plant defense, our results indicate that misregulated induction of phenylpropanoid metabolism also can be used to promote pathogen virulence.

Effects of water availability on emerald ash borer larval performance and phloem phenolics of Manchurian and black ash.

The invasive emerald ash borer (EAB) beetle is a significant threat to the survival of North American ash. In previous work, we identified putative biochemical and molecular markers of constitutive EAB resistance in Manchurian ash, an Asian species co-evolved with EAB. Here, we employed high-throughput high-performance liquid chromatography with photodiode array detection and mass spectrometry (HPLC-PDA-MS) to characterize the induced response of soluble phloem phenolics to EAB attack in resistant Manchurian and susceptible black ash under conditions of either normal or low water availability, and the effects of water availability on larval performance. Total larval mass per tree was lower in Manchurian than in black ash. Low water increased larval numbers and mean larval mass overall, but more so in Manchurian ash. Low water did not affect levels of phenolics in either host species, but six phenolics decreased in response to EAB. In both ashes, pinoresinol A was induced by EAB, especially in Manchurian ash. Pinoresinol A and pinoresinol B were negatively correlated with each other in both species. The higher accumulation of pinoresinol A in Manchurian ash after attack may help explain the resistance of this species to EAB, but none of the responses measured here could explain increased larval performance in trees subjected to low water availability.

Distinct roles for DNA-PK, ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress.

DNA damage encountered by DNA replication forks poses risks of genome destabilization, a precursor to carcinogenesis. Damage checkpoint systems cause cell cycle arrest, promote repair and induce programed cell death when damage is severe. Checkpoints are critical parts of the DNA damage response network that act to suppress cancer. DNA damage and perturbation of replication machinery causes replication stress, characterized by accumulation of single-stranded DNA bound by replication protein A (RPA), which triggers activation of ataxia telangiectasia and Rad3 related (ATR) and phosphorylation of the RPA32, subunit of RPA, leading to Chk1 activation and arrest. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [a kinase related to ataxia telangiectasia mutated (ATM) and ATR] has well characterized roles in DNA double-strand break repair, but poorly understood roles in replication stress-induced RPA phosphorylation. We show that DNA-PKcs mutant cells fail to arrest replication following stress, and mutations in RPA32 phosphorylation sites targeted by DNA-PKcs increase the proportion of cells in mitosis, impair ATR signaling to Chk1 and confer a G2/M arrest defect. Inhibition of ATR and DNA-PK (but not ATM), mimic the defects observed in cells expressing mutant RPA32. Cells expressing mutant RPA32 or DNA-PKcs show sustained H2AX phosphorylation in response to replication stress that persists in cells entering mitosis, indicating inappropriate mitotic entry with unrepaired damage.

Interspecific comparison of constitutive ash phloem phenolic chemistry reveals compounds unique to manchurian ash, a species resistant to emerald ash borer.

The emerald ash borer (Agrilus planipennis, EAB) is an invasive wood-borer indigenous to Asia and is responsible for widespread ash (Fraxinus spp.) mortality in the U.S. and Canada. Resistance and susceptibility to EAB varies among Fraxinus spp., which is a result of their co-evolutionary history with the pest. We characterized constitutive phenolic profiles and lignin levels in the phloem of green, white, black, blue, European, and Manchurian ash. Phloem was sampled twice during the growing season, coinciding with phenology of early and late instar EAB. We identified 66 metabolites that displayed a pattern of variation, which corresponded strongly with phylogeny. Previously identified lignans and lignan derivatives were confirmed to be unique to Manchurian ash, and may contribute to its high level of resistance to EAB. Other compounds that had been considered unique to Manchurian ash, including hydroxycoumarins and the phenylethanoids calceolarioside A and B, were detected in closely related, but susceptible species, and thus are unlikely to contribute to EAB resistance of Manchurian ash. The distinct phenolic profile of blue ash may contribute to its relatively high resistance to EAB.

Pollutants enhance IgE sensitization in the gut via local alteration of vitamin D-metabolizing enzymes.

Mechanisms linking ingested pollutants to increased incidence of allergy are poorly understood. We report that mice exposed to low doses of cadmium develop higher IgE responses following oral allergen sensitization and more severe allergic symptoms upon allergen challenge. The environmentally relevant doses of this pollutant also induced oxidative/inflammatory responses in the gut of SPF, but not germ-free mice. Interestingly, the increased IgE responses correlated with stimulation of the vitamin D3-metabolizing enzymes CYP27B1 and CYP24A1 in the gut and increased luminal levels of oxidized vitamin D3 metabolites that are not ligands of the vitamin D receptor. Inhibition of CYP27B1 and CYP24A1 via oral administration of pharmacological inhibitors reduced IgE responses induced in mice orally exposed to cadmium. Our findings identify local alteration of vitamin D signaling as a new mechanism for induction of IgE responses by environmental pollutants. They also identify vitamin D3-metabolizing enzymes as therapeutic targets for the treatment of allergy.

Evolution of the Kdo2-lipid A biosynthesis in bacteria.

BACKGROUND: Lipid A is the highly immunoreactive endotoxic center of lipopolysaccharide (LPS). It anchors the LPS into the outer membrane of most Gram-negative bacteria. Lipid A can be recognized by animal cells, triggers defense-related responses, and causes Gram-negative sepsis. The biosynthesis of Kdo2-lipid A, the LPS substructure, involves with nine enzymatic steps. RESULTS: In order to elucidate the evolutionary pathway of Kdo2-lipid A biosynthesis, we examined the distribution of genes encoding the nine enzymes across bacteria. We found that not all Gram-negative bacteria have all nine enzymes. Some Gram-negative bacteria have no genes encoding these enzymes and others have genes only for the first four enzymes (LpxA, LpxC, LpxD, and LpxB). Among the nine enzymes, five appeared to have arisen from three independent gene duplication events. Two of such events happened within the Proteobacteria lineage, followed by functional specialization of the duplicated genes and pathway optimization in these bacteria. CONCLUSIONS: The nine-enzyme pathway, which was established based on the studies mainly in Escherichia coli K12, appears to be the most derived and optimized form. It is found only in E. coli and related Proteobacteria. Simpler and probably less efficient pathways are found in other bacterial groups, with Kdo2-lipid A variants as the likely end products. The Kdo2-lipid A biosynthetic pathway exemplifies extremely plastic evolution of bacterial genomes, especially those of Proteobacteria, and how these mainly pathogenic bacteria have adapted to their environment.

A countrywide molecular survey leads to a seminal identification of the invasive cattle tick Rhipicephalus (Boophilus) microplus in Cameroon, a decade after it was reported in Cote d'Ivoire.

The cattle tick Rhipicephalus microplus is the most important arthropod vector of livestock diseases globally. Since its introduction in West Africa a decade ago, it has been reported in Ivory Coast, Benin, Togo, Mali, Burkina Faso and Nigeria with potentially far-reaching adverse impacts on the livestock sector in the region. Cameroon is located on a major route for transboundary cattle trade between Central and West Africa and it is therefore at risk from R. microplus invasion. This study investigated the occurrence of R. microplus in Cameroon, the genetic polymorphism of the tick and population structure of isolates from different regions of the country to provide data that underpin the design of future vector control programs. A cross-sectional survey was conducted in which ticks were collected from cattle at 54 sites across the five Agroecological zones (AEZs) within Cameroon. Tick identity (sex and species) was assigned using taxonomic keys. Species identity was confirmed through amplification and sequencing of the mitochondrial COI and 16S rRNA genes. A total of 7091 ticks were collected out of which 1112 (15.6%) were morphologically identified as R. microplus. The presence of R. microplus was confirmed in 4 out of 5 agroecological zones. Only two haplotypes were identified by both COI and 16S rRNA genes, indicating a very low divergence in the genetic structure of the R. microplus population in Cameroon. 16S rRNA sequence analysis revealed a new haplotype specific to Cameroon. Phylogenetic trees revealed that all isolates of R. microplus from Cameroon were grouped into the previously described Africa/Americas clade. Application of a niche modelling algorithm to R. microplus distribution in Cameroon predicted that suitable habitat for the tick extended into southern Nigeria. This study demonstrated for the first time the presence of R. microplus in Cameroon. Genetic diversity tests indicate that the tick has not evolved significantly since the initial introduction to West Africa. We suggest further longitudinal studies to better define the spatial and temporal expansion of the range of the tick and the drivers of this spread.

Application of alignment-free bioinformatics methods to identify an oomycete protein with structural and functional similarity to the bacterial AvrE effector protein.

Diverse plant pathogens export effector proteins to reprogram host cells. One of the most challenging goals in the molecular plant-microbe field is to functionally characterize the complex repertoires of effectors secreted by these pathogens. For bacterial pathogens, the predominant class of effectors is delivered to host cells by Type III secretion. For oomycetes, the predominant class of effectors is defined by a signal peptide that mediates secretion from the oomycete and a conserved RxLR motif. Downy mildew pathogens and Phytophthora species maintain hundreds of candidate RxLR effector genes in their genomes. Although no primary sequence similarity is evident between bacterial Type III effectors (T3Es) and oomycete RXLR effectors, some bacterial and oomycete effectors have convergently evolved to target the same host proteins. Such effectors might have evolved domains that are functionally similar but sequence-unrelated. We reasoned that alignment-free bioinformatics approaches could be useful to identify structural similarities between bacterial and oomycete effectors. To test this approach, we used partial least squares regression, alignment-free bioinformatics methods to identify effector proteins from the genome of the oomycete Hyaloperonospora arabidopsidis that are similar to the well-studied AvrE1 effector from Pseudomonas syringae. This approach identified five RxLR proteins with putative structural similarity to AvrE1. We focused on one, HaRxL23, because it is an experimentally validated effector and it is conserved between distantly related oomycetes. Several experiments indicate that HaRxL23 is functionally similar to AvrE1, including the ability to partially rescue an AvrE1 loss-of-function mutant. This study provides an example of how an alignment-free bioinformatics approach can identify functionally similar effector proteins in the absence of primary sequence similarity. This approach could be useful to identify effectors that have convergently evolved regardless of whether the shared host target is known.

Fecal microbiome of periparturient dairy cattle and associations with the onset of Salmonella shedding.

Non-typhoidal Salmonella enterica is a zoonotic pathogen with critical importance in animal and public health. The persistence of Salmonella on farms affects animal productivity and health, and represents a risk for food safety. The intestinal microbiota plays a fundamental role in the colonization and invasion of this ubiquitous microorganism. To overcome the colonization resistance imparted by the gut microbiome, Salmonella uses invasion strategies and the host inflammatory response to survive, proliferate, and establish infections with diverse clinical manifestations. Cattle serve as reservoirs of Salmonella, and periparturient cows have high prevalence of Salmonella shedding; however, little is known about the association between the gut microbiome and the onset of Salmonella shedding during the periparturient period. Thus, the objective of this study was to assess the association between changes in bacterial communities and the onset of Salmonella shedding in cattle approaching parturition. In a prospective cohort study, fecal samples from 98 dairy cows originating from four different farms were collected at four time points relative to calving (-3 wks, -1 wk, +1 wk, +3 wks). All 392 samples were cultured for Salmonella. Sequencing of the V4 region of the 16S rRNA gene using the Illumina platform was completed to evaluate the fecal microbiome in a selected sample subset. Analyses of microbial composition, diversity, and structure were performed according to time points, farm, and Salmonella onset status. Individual cow fecal microbiomes, predominated by Bacteroidetes, Firmicutes, Spirochaetes, and Proteobacteria phyla, significantly changed before and after parturition. Microbial communities from different farms were distinguishable based on multivariate analysis. Although there were significant differences in some bacterial taxa between Salmonella positive and negative samples, our results did not identify differences in the fecal microbial diversity or structure for cows with and without the onset of Salmonella shedding. These data suggest that determinants other than the significant changes in the fecal microbiome influence the periparturient onset of Salmonella shedding in dairy cattle.

DNA-PK phosphorylation of RPA32 Ser4/Ser8 regulates replication stress checkpoint activation, fork restart, homologous recombination and mitotic catastrophe.

Genotoxins and other factors cause replication stress that activate the DNA damage response (DDR), comprising checkpoint and repair systems. The DDR suppresses cancer by promoting genome stability, and it regulates tumor resistance to chemo- and radiotherapy. Three members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, ATM, ATR, and DNA-PK, are important DDR proteins. A key PIKK target is replication protein A (RPA), which binds single-stranded DNA and functions in DNA replication, DNA repair, and checkpoint signaling. An early response to replication stress is ATR activation, which occurs when RPA accumulates on ssDNA. Activated ATR phosphorylates many targets, including the RPA32 subunit of RPA, leading to Chk1 activation and replication arrest. DNA-PK also phosphorylates RPA32 in response to replication stress, and we demonstrate that cells with DNA-PK defects, or lacking RPA32 Ser4/Ser8 targeted by DNA-PK, confer similar phenotypes, including defective replication checkpoint arrest, hyper-recombination, premature replication fork restart, failure to block late origin firing, and increased mitotic catastrophe. We present evidence that hyper-recombination in these mutants is ATM-dependent, but the other defects are ATM-independent. These results indicate that DNA-PK and ATR signaling through RPA32 plays a critical role in promoting genome stability and cell survival in response to replication stress.

Nutritional attributes of ash (Fraxinus spp.) outer bark and phloem and their relationships to resistance against the emerald ash borer.

The emerald ash borer (Agrilus planipennis Fairmaire, EAB) is an alien, invasive wood-boring insect that is responsible for killing millions of ash trees since its discovery in North America in 2002. All North American ash species (Fraxinus spp.) that EAB has encountered have shown various degrees of susceptibility, while Manchurian ash (Fraxinus mandshurica Ruprecht), which shares a co-evolutionary history with this insect, is resistant. Recent studies have looked into constitutive resistance mechanisms in Manchurian ash, concentrating on the secondary phloem, which is the feeding substrate for the insect. In addition to specialized metabolism and defense-related components, primary metabolites and nutritional summaries can also be important to understand the feeding behavior of insect herbivores. Here, we have compared the nutritional characteristics (water content, total protein, free amino acids, total soluble sugars and starch, percent carbon and nitrogen, and macro- and micronutrients) of outer bark and phloem from black, green, white and Manchurian ash to determine their relevance to resistance or susceptibility to EAB. Water content and concentrations of Al, Ba, Cu, Fe, K, Li, tryptophan and an unknown compound were found to separate black and Manchurian ash from green and white ash in a principal component analysis (PCA), confirming their phylogenetic placements into two distinct clades. The traits that distinguished Manchurian ash from black ash in the PCA were water content and concentrations of total soluble sugars, histidine, lysine, methionine, ornithine, proline, sarcosine, tyramine, tyrosol, Al, Fe, K, Na, V and an unknown compound. However, only proline, tyramine and tyrosol were significantly different, and higher, in Manchurian ash than in black ash.

RNA-Seq analysis of the Sclerotinia homoeocarpa--creeping bentgrass pathosystem.

Sclerotinia homoeocarpa causes dollar spot disease, the predominate disease on highly-maintained turfgrass. Currently, there are major gaps in our understanding of the molecular interactions between S. homoeocarpa and creeping bentgrass. In this study, 454 sequencing technology was used in the de novo assembly of S. homoeocarpa and creeping bentgrass transcriptomes. Transcript sequence data obtained using Illumina's first generation sequencing-by-synthesis (SBS) were mapped to the transcriptome assemblies to estimate transcript representation in different SBS libraries. SBS libraries included a S. homoeocarpa culture control, a creeping bentgrass uninoculated control, and a library for creeping bentgrass inoculated with S. homoeocarpa and incubated for 96 h. A Fisher's exact test was performed to determine transcripts that were significantly different during creeping bentgrass infection with S. homoeocarpa. Fungal transcripts of interest included glycosyl hydrolases, proteases, and ABC transporters. Of particular interest were the large number of glycosyl hydrolase transcripts that target a wide range of plant cell wall compounds, corroborating the suggested wide host range and saprophytic abilities of S. homoeocarpa. Several of the multidrug resistance ABC transporters may be important for resistance to both fungicides and plant defense compounds. Creeping bentgrass transcripts of interest included germins, ubiquitin transcripts involved in proteasome degradation, and cinnamoyl reductase, which is involved in lignin production. This analysis provides an extensive overview of the S. homoeocarpa-turfgrass pathosystem and provides a starting point for the characterization of potential virulence factors and host defense responses. In particular, determination of important host defense responses may assist in the development of highly resistant creeping bentgrass varieties.

Mining Cytochrome b561 proteins from plant genomes.

Cytochrome b561 (Cyt-b561) proteins are important for plant growth, development, and prevention of damage to plants. Because of their high sequence divergence, thorough mining of Cyt-b561 proteins from plant genomes are not easy. Currently there is only one Cyt-b561 gene found in the maize and none in the soybean genome. However, 22 have been identified in the Arabidopsis thaliana genome. We tested alignment-free protein classifiers based on partial least squares (PLS) and support vector machines to identify Cyt-b561. These classifiers performed better than profile hidden Markov models and PSI-BLAST. Using these classifiers we identified new Cyt-b561-related proteins from four plant genomes.

Protein family classification with partial least squares.

The quality of protein function predictions relies on appropriate training of protein classification methods. Performance of these methods can be affected when only a limited number of protein samples are available, which is often the case in divergent protein families. Whereas profile hidden Markov models and PSI-BLAST presented significant performance decrease in such cases, alignment-free partial least-squares classifiers performed consistently better even when used to identify short fragmented sequences.

Mining the Arabidopsis thaliana genome for highly-divergent seven transmembrane receptors.

To identify divergent seven-transmembrane receptor (7TMR) candidates from the Arabidopsis thaliana genome, multiple protein classification methods were combined, including both alignment-based and alignment-free classifiers. This resolved problems in optimally training individual classifiers using limited and divergent samples, and increased stringency for candidate proteins. We identified 394 proteins as 7TMR candidates and highlighted 54 with corresponding expression patterns for further investigation.