RNA silencing proteins and small RNAs in oomycete plant pathogens and biocontrol agents.

Introduction: Oomycetes cause several damaging diseases of plants and animals, and some species also act as biocontrol agents on insects, fungi, and other oomycetes. RNA silencing is increasingly being shown to play a role in the pathogenicity of Phytophthora species, either through trans-boundary movement of small RNAs (sRNAs) or through expression regulation of infection promoting effectors. Methods: To gain a wider understanding of RNA silencing in oomycete species with more diverse hosts, we mined genome assemblies for Dicer-like (DCL), Argonaute (AGO), and RNA dependent RNA polymerase (RDRP) proteins from Phytophthora plurivora, Ph. cactorum, Ph. colocasiae, Pythium oligandrum, Py. periplocum, and Lagenidium giganteum. Moreover, we sequenced small RNAs from the mycelium stage in each of these species. Results and discussion: Each of the species possessed a single DCL protein, but they differed in the number and sequence of AGOs and RDRPs. SRNAs of 21nt, 25nt, and 26nt were prevalent in all oomycetes analyzed, but the relative abundance and 5' base preference of these classes differed markedly between genera. Most sRNAs mapped to transposons and other repeats, signifying that the major role for RNA silencing in oomycetes is to limit the expansion of these elements. We also found that sRNAs may act to regulate the expression of duplicated genes. Other sRNAs mapped to several gene families, and this number was higher in Pythium spp., suggesting a role of RNA silencing in regulating gene expression. Genes for most effector classes were the source of sRNAs of variable size, but some gene families showed a preference for specific classes of sRNAs, such as 25/26 nt sRNAs targeting RxLR effector genes in Phytophthora species. Novel miRNA-like RNAs (milRNAs) were discovered in all species, and two were predicted to target transcripts for RxLR effectors in Ph. plurivora and Ph. cactorum, indicating a putative role in regulating infection. Moreover, milRNAs from the biocontrol Pythium species had matches in the predicted transcriptome of Phytophthora infestans and Botrytis cinerea, and L. giganteum milRNAs matched candidate genes in the mosquito Aedes aegypti. This suggests that trans-boundary RNA silencing may have a role in the biocontrol action of these oomycetes.

Severe meningoencephalitis secondary to calvarial invasion of Lagenidium giganteum forma caninum in a dog.

Background: The oomycete Lagenidiumgiganteum forma caninum is an uncommon cause of severe dermal and subcutaneous infections in dogs with possible vascular invasion and other fatal sequelae. Infection within the central nervous system of affected dogs has not been previously reported. Case Description: A 6-year-old spayed female mixed-breed dog was evaluated at a referral institution with a 2-month history of suspected fungal infection in the region of the right mandibular lymph node that was refractory to surgical resection and empiric medical therapy. Physical examination identified a 6-cm fluctuant subcutaneous mass caudoventral to the ramus of the right mandible and a second firm mass in the region of the right caudal maxilla. Lesional punch biopsies were submitted for fungal culture and polymerase chain reaction (PCR), which subsequently identified L. giganteum forma caninum infection. Initial treatment consisted of anti-inflammatory doses of prednisone and hyperbaric oxygen therapy. Four weeks following initial evaluation, the patient was presented with progressive neurological signs consistent with a forebrain lesion. Magnetic resonance imaging revealed soft-tissue, contrast-enhancing lesions ventral to the calvarium adjacent to the site of original surgical resection and throughout the brain. Humane euthanasia was elected, and postmortem examination was consistent with the extension of local disease from the right masseter muscle into the right ventral calvarium. Postmortem DNA sequencing confirmed the identity of the organism as L. giganteum forma caninum. Conclusion: This is the first reported case of intracranial lagenidiosis in the dog. PCR distinguished this species from other Lagenidium species and from oomycetes of other genera, such as Pythiuminsidiosum and Paralagenidium karlingii. Regional extension of cutaneous lagenidiosis should therefore be considered in cases with concurrent or spontaneous neurologic disease.

Glycoside hydrolases family 20 (GH20) represent putative virulence factors that are shared by animal pathogenic oomycetes, but are absent in phytopathogens.

BACKGROUND: Although interest in animal pathogenic oomycetes is increasing, the molecular basis mediating oomycete-animal relationships remains virtually unknown. Crinkler (CRN) genes, which have been traditionally associated with the cytotoxic activity displayed by plant pathogenic oomycetes, were recently detected in transcriptome sequences from the entomopathogenic oomycete Lagenidium giganteum, suggesting that these genes may represent virulence factors conserved in both animal and plant pathogenic oomycetes. In order to further characterize the L. giganteum pathogenome, an on-going genomic survey was mined to reveal novel putative virulence factors, including canonical oomycete effectors Crinkler 13 (CRN13) orthologs. These novel sequences provided a basis to initiate gene expression analyses and determine if the proposed L. giganteum virulence factors are differentially expressed in the presence of mosquito larvae (Aedes aegypti). RESULTS: Sequence analyses revealed that L. giganteum express CRN13 transcripts. The predicted proteins, like other L. giganteum CRNs, contained a conserved LYLA motif at the N terminal, but did not display signal peptides. In contrast, other potential virulence factors, such as Glycoside Hydrolases family 20 (hexosaminidase) and 37 (trehalase) proteins (GH20 and GH37), contained identifiable signal peptides. Genome mining demonstrated that GH20 genes are absent from phytopathogenic oomycete genomes, and that the L. giganteum GH20 sequence is the only reported peronosporalean GH20 gene. All other oomycete GH20 homologs were retrieved from animal pathogenic, saprolegnialean genomes. Furthermore, phylogenetic analyses demonstrated that saprolegnialean and peronosporalean GH20 protein sequences clustered in unrelated clades. The saprolegnialean GH20 sequences appeared as a strongly supported, monophyletic group nested within an arthropod-specific clade, suggesting that this gene was acquired via a lateral gene transfer event from an insect or crustacean genome. In contrast, the L. giganteum GH20 protein sequence appeared as a sister taxon to a plant-specific clade that included exochitinases with demonstrated insecticidal activities. Finally, gene expression analyses demonstrated that the L. giganteum GH20 gene expression level is significantly modulated in the presence of mosquito larvae. In agreement with the protein secretion predictions, CRN transcripts did not show any differential expression. CONCLUSIONS: These results identified GH20 enzymes, and not CRNs, as potential pathogenicity factors shared by all animal pathogenic oomycetes.

Change of Lipid,Esterase and Lipase in Mosquito Larvae Infected with Lagenidium giganteum / 中国寄生虫学与寄生虫病杂志

Objective To explore possible physiological and biochemical mechanisms of Lagenidium giganteum infection in killing mosquito larvae. Methods The content of lipid and the activities of esterase and lipase between the normal and infected mosquito larvae were observed with histochemical method. The results were photomicrographed and analysed by image analysis using computer. Results In 24 hrs after infection, the content of lipid in the infected Culex quinquefasciatus larvae was lower while the activities of esterase and lipase in the larvae were higher than the control. In 48 hrs and 78 hrs after infection, the content of lipid in the infected larvae of C. quinquefasciatus and Ae. albopictus were significantly lower while the activities of esterase and lipase were significantly higher than those of control. Conclusion The content of lipid in the mosquito larvae decreased while the activities of esterase and lipase increased after the infection of Lagenidium giganteum . The disorder of lipid metabolism might be part of the killing mechanisms to C. quinquefasciatus and Ae. albopictus larvae.

EFFECTS OF LAGENIDIUM GIGANTEUM ON THE ACTIVITIES OF THREE ENZYMES OF CULEX QUINQUEFASCIATUS LARVAE / 中国寄生虫学与寄生虫病杂志

Objective] To investigate the possible mechanism underlying the killing of Culex quinquefasciatus larvae by Lagenidium giganteum from the biochemical point of view. [Methods] The activities of alkaline phosphatase(AKP), acid phosphatase(ACP) and esterase(EST) were observed by using calcium cobalt method, lead nitrate method and naphthol method. Results were photomicrographed and analysed quantitatively by image analysis using computer. [Results] The activities of AKP and ACP were decreased while the activities of EST were increased among the infected groups with different infection intensities. [Conclusions] The changes of these enzyme activities might be one of the important mechanisms of action of Lagenidium giganteum against Culex quinquefasciafus larvae.

Changes of Elements in Culex quinquefasciatus Larvae after Infected by Lagenidium giganteum (Oomycetes:lagenidiales) / 中国寄生虫学与寄生虫病杂志

Objective To analyze the quantitative change of 18 elements in Culex quinquefasciatus Larvae infected by Lagenidium giganteum (Oomycetes:lagenidiales), and to explore possible mechanism of mosquito-killing by the fungus. Methods. Elements in the mosquito larvae at different developmental stages from infected and non-infected (control) groups were detected with automatic bioassay machine. Results . At the early stage of infection, content of Ca and Cu in the infected group was markedly lower while the content of Co and Cd were higher than the control. In 24 hrs after infection, the content of Ca, Mg, Na, P, Fe, Zn, Cu, Mn and Cr was significantly lower while that of K, Mo, Co, Ni, Al, Cd and Pb were higher than those in non-infected larvae. No marked difference was found for the content of Sr and B between the two groups. In 48 hrs after infection, considerable difference was found in 17 elements between infected and non-infected larvae, with only one exception of Sr. Conclusion . Many elements which play an important role in maintaining the vitality of the mosquito larvae decreased and some of the heavy metal elements accumulated after the infection of Lagenidium giganteum. This might be a part of the killing mechanism to Culex quinquefasciatus larvae.