Host‐ and stage‐dependent secretome of the arbuscular mycorrhizal fungus Rhizophagus irregularis

Arbuscular mycorrhizal fungi form the most wide‐spread endosymbiosis with plants. There is very little host specificity in this interaction, however host preferences as well as varying symbiotic efficiencies have been observed. We hypothesize that secreted proteins (SPs) may act as fungal effectors to control symbiotic efficiency in a host‐dependent manner. Therefore, we studied whether arbuscular mycorrhizal (AM) fungi adjust their secretome in a host‐ and stage‐dependent manner to contribute to their extremely wide host range. We investigated the expression of SP‐encoding genes of Rhizophagus irregularis in three evolutionary distantly related plant species, Medicago truncatula, Nicotiana benthamiana and Allium schoenoprasum. In addition we used laser microdissection in combination with RNA‐seq to study SP expression at different stages of the interaction in Medicago. Our data indicate that most expressed SPs show roughly equal expression levels in the interaction with all three host plants. In addition, a subset shows significant differential expression depending on the host plant. Furthermore, SP expression is controlled locally in the hyphal network in response to host‐dependent cues. Overall, this study presents a comprehensive analysis of the R. irregularis secretome, which now offers a solid basis to direct functional studies on the role of fungal SPs in AM symbiosis.     

Molecular phylogeny of Osmanthus (Oleaceae) based on non‐coding chloroplast and nuclear ribosomal internal transcribed spacer regions

Abstract The phylogenetic relationships of Osmanthus Lour. were investigated using the nuclear ribosomal internal transcribed spacer (ITS) regions and non‐coding chloroplast regions (psbA‐trnH, trnL‐F). The two datasets support the conclusion that Osmanthus is polyphyletic, with some species of the subtribe Oleinae nested within Osmanthus. Osmanthus didymopetalus P. S. Green is nested within the clade formed by species of section Osmanthus in two trees. Osmanthus attenuatus P. S. Green, O. yunnanensis P. S. Green, and O. gracilinervis R. L. Lu of traditional section Osmanthus are clearly divergent from other accessions, and do not form a monophyletic group with other Osmanthus accessions. Osmanthus marginatus Hemsl. is embedded in the clade formed by species of section Osmanthus in the ITS tree. In cpDNA trees all species of section Osmanthus are placed in the large clade and all species of section Leiolea formed a group. The taxonomic incongruence among trees for ITS and cpDNA indicate hybridization, as introgression may have occurred among some species of sections Osmanthus and Leiolea. Phylogeny of Osmanthus is discussed in light of molecular and morphological data, and a revised infrageneric classification with three sections (Leiolea, Siphosmanthu, and Osmanthus) is presented. The section Linocieroides is abandoned and united with section Osmanthus.     

Sequence differences in the internal transcribed spacer ribosomal DNA among four species of hookworm (Ancylostomatoidea: Ancylostoma)

The two ribosomal DNA internal transcribed spacers (1 and 2) of the hookworms Ancylostoma caninum, A. tubaeforme, A. ceylanicum and A. duodenale were sequenced. The sequence lengths were similar among the four species, except that A. ceylanicum had slightly longer (by 5–7 bp) internal transcribed spacer 1 and 2 sequences. The predicted secondary structure of the internal transcribed spacer 2 precursor rRNA was similar for all species, despite interspecific differences in primary sequence ranging from 0.9% to 13.2%. Interspecific differences in internal transcribed spacer 1 sequence ranged from 0.9% to 7.5%. A cladistic analysis of the sequence data, using the human hookworm Necator americanus as the outgroup, provided little resolution of the phylogenetic relationships, except that A. ceylanicum occurred on a branch external to the other three species. Nonetheless, internal transcribed spacers 1 and 2 may provide useful phylogenetic information at higher taxonomic levels within the superfamily Ancylostomatoidea.     

Effect of swine leukocyte antigen‐DQA gene variation on diarrhea in Large White,Landrace, and Duroc piglets

Piglet diarrhea is one of the most common factors that affects the benefits of the swine industry. Although recent studies have shown that exon 2 of SLA‐DQA is associated with piglet resistance to diarrhea, contributions of genetic variation in the additional exon coding regions of this gene remain unclear. Here, we investigated variation in exons 1, 3 and 4 of the SLA‐DQA gene and evaluated their effects on diarrheal infection in 425 suckling piglets. No variation was identified in exon 1. In exon 3, there were eight alleles detected, generated by 14 single nucleotide polymorphisms (SNPs) and three nucleotide deletions, eight SNPs being newly identified. Four allele sequences and three SNPs were identified in exon 4, only one SNP being newly identified. Statistical analysis showed that the genotypes of exon 3 are significantly associated with piglet diarrhea; indeed, genotypes DQA*wb01/wb02 and wb04/wb05 are clearly associated with resistance to piglet diarrhea, as they have the lowest probabilities of infection (P < 0.05). However, no significant association was found between the genotypes of exon 4 and diarrhea (P > 0.05). These results provide important new information concerning the level of genetic diversity at the SLA‐DQA locus and suggest that further genetic association studies of piglet diarrhea resistance should include analyses of both exons 2 and 3 of this locus.     

Genetic variation in 16S-23S rDNA internal transcribed spacer regions and the possible use of this genetic variation for molecular diagnosis of Bacteroides species

The structural variation in 16S-23S rDNA internal transcribed spacer regions (ITS) among Bacteroides species was assessed by PCR amplification and sequencing analysis, and its possible use for molecular diagnosis of these species was evaluated. Ninety strains of the genus Bacteroides, including the species B. distasonis, B. eggerthii, B. fragilis, B. ovatus, B. thetaiotaomicron, B. uniformis and B. vulgatus, produced one to three ITS amplification products with sizes ranging from 615 to 810 bp. Some Bacteroides strains could be differentiated at species level on the basis of ITS amplification patterns and restriction fragment length polymorphism (RFLP) analysis using a four-nucleotide-recognizing enzyme, Msp I. The results of sequence analysis of ITS amplification products revealed genes for Ile-tRNA and Ala-tRNA in all strains tested. The nucleotide sequence, except for that in tRNA-coding regions, was highly variable and characteristic for each species, but a common sequence among B. fragilis, B. thetaiotaomicron and B. ovatus was observed. A digoxigenin-labeled oligonucleotide probe (named FOT1), which was designed from this conserved sequence, specifically hybridized to the ITS amplification products from B. fragilis, B. thetaiotaomicron and B. ovatus. These results suggest that the ITS region is a useful target for the development of rapid and accurate techniques for identification of Bacteroides species.     

GTSF‐1 is required for formation of a functional RNA‐dependent RNA Polymerase complex in Caenorhabditis elegans

Argonaute proteins and their associated small RNAs (sRNAs) are evolutionarily conserved regulators of gene expression. Gametocyte‐specific factor 1 (Gtsf1) proteins, characterized by two tandem CHHC zinc fingers and an unstructured C‐terminal tail, are conserved in animals and have been shown to interact with Piwi clade Argonautes, thereby assisting their activity. We identified the Caenorhabditis elegans Gtsf1 homolog, named it gtsf‐1 and characterized it in the context of the sRNA pathways of C. elegans. We report that GTSF‐1 is not required for Piwi‐mediated gene silencing. Instead, gtsf‐1 mutants show a striking depletion of 26G‐RNAs, a class of endogenous sRNAs, fully phenocopying rrf‐3 mutants. We show, both in vivo and in vitro, that GTSF‐1 interacts with RRF‐3 via its CHHC zinc fingers. Furthermore, we demonstrate that GTSF‐1 is required for the assembly of a larger RRF‐3 and DCR‐1‐containing complex (ERIC), thereby allowing for 26G‐RNA generation. We propose that GTSF‐1 homologs may act to drive the assembly of larger complexes that act in sRNA production and/or in imposing sRNA‐mediated silencing activities.     

A pair of receptor‐like kinases is responsible for natural variation in shoot growth response to mannitol treatment in Arabidopsis thaliana

Growth is a complex trait that adapts to the prevailing conditions by integrating many internal and external signals. Understanding the molecular origin of this variation remains a challenging issue. In this study, natural variation of shoot growth under mannitol‐induced stress was analyzed by standard quantitative trait locus mapping methods in a recombinant inbred line population derived from a cross between the Col‐0 and Cvi‐0 Arabidopsis thaliana accessions. Cloning of a major QTL specific to mannitol‐induced stress condition led to identification of EGM1 and EGM2, a pair of tandem‐duplicated genes encoding receptor‐like kinases that are potentially involved in signaling of mannitol‐associated stress responses. Using various genetic approaches, we identified two non‐synonymous mutations in the EGM2[Cvi] allele that are shared by at least ten accessions from various origins and are probably responsible for a specific tolerance to mannitol. We have shown that the enhanced shoot growth phenotype contributed by the Cvi allele is not linked to generic osmotic properties but instead to a specific chemical property of mannitol itself. This result raises the question of the function of such a gene in A. thaliana, a species that does not synthesize mannitol. Our findings suggest that the receptor‐like kinases encoded by EGM genes may be activated by mannitol produced by pathogens such as fungi, and may contribute to plant defense responses whenever mannitol is present.     

Resistance and resilience of root fungal communities to water limitation in a temperate agroecosystem

Understanding crop resilience to environmental stress is critical in predicting the consequences of global climate change for agricultural systems worldwide, but to date studies addressing crop resiliency have focused primarily on plant physiological and molecular responses. Arbuscular mycorrhizal fungi (AMF) form mutualisms with many crop species, and these relationships are key in mitigating the effects of abiotic stress in many agricultural systems. However, to date there is little research examining whether (1) fungal community structure in agroecosystems is resistant to changing environmental conditions, specifically water limitation and (2) resilience of fungal community structure is moderated by agricultural management systems, namely the integration of trees into cropping systems. Here, we address these uncertainties through a rainfall reduction field experiment that manipulated short‐term water availability in a soybean‐based (Glycine max L. Merr.) agroforest in Southern Ontario, Canada. We employed terminal restriction fragment length polymorphism analysis to determine the molecular diversity of both general fungal and AMF communities in soybean roots under no stress, stress (rainfall shelters added), and poststress (rainfall shelters removed). We found that general fungal and AMF communities sampled from soybean roots were resistant to rainfall reduction in a monoculture, but not in an agroforest. While AMF communities were unchanged after stress removal, general fungal communities were significantly different poststress in the agroforest, indicating a capacity for resiliency. Our study indicates that generalist fungi and AMF are responsive to changes in environmental conditions and that agroecosystem management plays a key role in the resistance and resilience of fungal communities to water limitation.     

Sequence variation in the cytochrome oxidase I, internal transcribed spacer 1, and Ts14 diagnostic antigen sequences of Taenia solium isolates from South and Central America, India, and Asia

We examined the genetic variability in the pig–human tapeworm, Taenia solium, by sequencing the genes for cytochrome oxidase I, internal transcribed spacer 1, and a diagnostic antigen, Ts14, from individual cysts isolated from Peru, Colombia, Mexico, India, China, and the Philippines. For these genes, the rate of nucleotide variation was minimal. Isolates from these countries can be distinguished based on one to eight nucleotide differences in the 396 nucleotide cytochrome oxidase I (COI) sequence. However, all of the 15 isolates from within Peru had identical COI sequences. The Ts14 sequences from India and China were identical and differed from the Peru sequence by three nucleotides in 333. These data indicate that there is minimal genetic variability within the species T. solium. Minimal variability was also seen in the ITS1 sequence, but this variation was observed within the individual. Twenty-two cloned sequences from six isolates sorted into 13 unique sequences. The variability observed within the sequences from individual cysts was as great as the variability between the isolates.     

Density‐dependent fitness,not dispersal movements,drives temporal variation in spatial genetic structure in dark‐eyed juncos (Junco hyemalis)

Some studies have found that dispersal rates and distances increase with density, indicating that density‐dependent dispersal likely affects spatial genetic structure. In an 11‐year mark–recapture study on a passerine, the dark‐eyed junco, we tested whether density affected dispersal distance and/or fine‐scale spatial genetic structure. Contrary to expectations, we found no effect of predispersal density on dispersal distance or the proportion of locally produced juveniles returning to the population from which they hatched. However, even though density did not affect dispersal distance or natal return rates, we found that density still did affect spatial genetic structure. We found significant positive spatial genetic structure at low densities of (postdispersal) adults but not at high densities. In years with high postdispersal (adult) densities that also had high predispersal (juvenile) densities in the previous year, we found negative spatial genetic structure, indicating high levels of dispersal. We found that density also affected fitness of recruits, and fitness of immigrants, potentially linking these population parameters with the spatial genetic structure detected. Immigrants and recruits rarely nested in low postdispersal density years. In contrast, in years with high postdispersal density, recruits were common and immigrants had equal success to local birds, so novel genotypes diluted the gene pool and effectively eliminated positive spatial genetic structure. In relation to fine‐scale spatial genetic structure, fitness of immigrants and new recruits is poorly understood compared to dispersal movements, but we conclude that it can have implications for the spatial distribution of genotypes in populations.     

Polyclonal antibody‐based ELISA in combination with specific PCR amplification of internal transcribed spacer regions for the detection and quantitation of Lasiodiplodia theobromae,causal agent of gummosis in cashew nut plants

Members of Botryosphaeriaceae family are associated with serious diseases in different plants across the world. In cashew nut plants (Anacardium occidentale), the fungus Lasiodiplodia theobromae causes a severe group of symptoms related to gummosis that results in decreased nut production. The aim of this work was to develop an indirect enzyme‐linked immunosorbent assay (ELISA) with sufficient sensitivity and specificity to detect the fungus both in vitro and in planta (artificially and naturally infected) and to increase the detection specificity within the fungi group using primers specific for the internal transcribed spacer (ITS) sequences. A collection of L. theobromae isolates was obtained, and antisera against the fungus were raised in rabbits. Cross‐reactivity against Neofusicoccum sp., Colletotrichum gloeosporioides, Phomopsis anacardii and Pestalotiopsis guepinii was examined. Naturally and artificially infected vegetal material were employed in the ELISAs. The fungi ITS sequences were determined, and single nucleotide polymorphisms were identified and used for primer design. For the naturally infected plants, there was an approximately fourfold variation in the absorbance values. Some positive readings for asymptomatic samples were detected. For the artificially infected samples, an ELISA‐based weekly time‐course analysis was conducted, and the values for samples from 0 and 7 days were lower than the threshold value. Beginning on day 14, the infection could be detected, with rates varying from 40% on day 14 to 80% on day 21 and 100% by the end of the experiment. The ITS sequencing revealed few polymorphisms among the L. theobromae isolates, but for C. gloeosporioides, P. anacardii, P. guepinii and Neofusicoccum sp., the sequences were sufficient to permit reliable discrimination. The feasibility of ELISA as an early detection technique to assist in gummosis management was demonstrated. PCR amplification based on ITS regions increases and complements serological specificity.     

Symbiont strain is the main determinant of variation in Wolbachia‐mediated protection against viruses across Drosophila species

Wolbachia is a common heritable bacterial symbiont in insects. Its evolutionary success lies in the diverse phenotypic effects it has on its hosts coupled to its propensity to move between host species over evolutionary timescales. In a survey of natural host–symbiont associations in a range of Drosophila species, we found that 10 of 16 Wolbachia strains protected their hosts against viral infection. By moving Wolbachia strains between host species, we found that the symbiont genome had a much greater influence on the level of antiviral protection than the host genome. The reason for this was that the level of protection depended on the density of the symbiont in host tissues, and Wolbachia rather than the host‐controlled density. The finding that virus resistance and symbiont density are largely under the control of symbiont genes in this system has important implications both for the evolution of these traits and for public health programmes using Wolbachia to prevent mosquitoes from transmitting disease.     

Sequence variation in the cytochrome oxidase I, internal transcribed spacer 1, and Ts14 diagnostic antigen sequences of Taenia solium isolates from South and Central America, India, and Asia.

We examined the genetic variability in the pig–human tapeworm, Taenia solium, by sequencing the genes for cytochrome oxidase I, internal transcribed spacer 1, and a diagnostic antigen, Ts14, from individual cysts isolated from Peru, Colombia, Mexico, India, China, and the Philippines. For these genes, the rate of nucleotide variation was minimal. Isolates from these countries can be distinguished based on one to eight nucleotide differences in the 396 nucleotide cytochrome oxidase I (COI) sequence. However, all of the 15 isolates from within Peru had identical COI sequences. The Ts14 sequences from India and China were identical and differed from the Peru sequence by three nucleotides in 333. These data indicate that there is minimal genetic variability within the species T. solium. Minimal variability was also seen in the ITS1 sequence, but this variation was observed within the individual. Twenty-two cloned sequences from six isolates sorted into 13 unique sequences. The variability observed within the sequences from individual cysts was as great as the variability between the isolates.