NbSOBIR1 Partitions Into Plasma Membrane Microdomains and Binds ER-Localized NbRLP1.

The receptor-like kinase Suppressor of BIR1 (SOBIR1) binds various receptor-like proteins (RLPs) that perceive microbe-associated molecular patterns (MAMPs) at the plasma membrane, which is thought to activate plant pattern-triggered immunity (PTI) against pathogen invasion. Despite its potentially crucial role, how SOBIR1 transmits immune signaling to ultimately elicit PTI remains largely unresolved. Herein, we report that a Nicotiana benthamiana gene NbRLP1, like NbSOBIR1, was highly induced upon Phytophthora parasitica infection. Intriguingly, NbRLP1 is characterized as a receptor-like protein localizing to the endoplasmic reticulum (ER) membrane rather than the plasma membrane. Using bimolecular fluorescence complementation and affinity purification assays, we established that NbRLP1 is likely to associate with NbSOBIR1 through the contact between the ER and plasma membrane. We further found that NbSOBIR1 at the plasma membrane partitions into mobile microdomains that undergo frequent lateral movement and internalization. Remarkably, the dynamics of NbSOBIR1 microdomain is coupled to the remodeling of the cortical ER network. When NbSOBIR1 microdomains were induced by the P. parasitica MAMP ParA1, tobacco cells overexpressing NbRLP1 accelerated NbSOBIR1 internalization. Overexpressing NbRLP1 in tobacco further exaggerated the ParA1-induced necrosis. Together, these findings have prompted us to propose that ER and the ER-localized NbRLP1 may play a role in transmitting plant immune signals by regulating NbSOBIR1 internalization.

Soil Is Not a Reservoir for Phellinus noxius.

Phellinus noxius causes brown root rot (BRR) of diverse trees. Basidiospores and diseased host tissues have been recognized as important sources of P. noxius inoculum. This study aimed to understand whether P. noxius could occur or survive in soil without host tissues in the natural environment. Soil was sampled before and after the removal of diseased trees at eight BRR infection sites (total of 44 samples). No P. noxius colonies were recovered in soil plating assays, suggesting that no or little viable P. noxius resided in the soil. To know whether P. noxius could disseminate from decayed roots to the surrounding soil, rhizosphere and non-rhizosphere soils were sampled from another two infection sites. Although P. noxius DNA was detectable with specific primers, no P. noxius could be isolated, even from the rhizosphere soils around decayed roots covered with P. noxius mycelial mats. The association between viable P. noxius and the presence of its DNA was also investigated using field soil mixed with P. noxius arthrospores. After P. noxius was exterminated by flooding or fumigation treatment, its DNA remained detectable for a few weeks. The potential of onsite soil as an inoculum was tested using the highly susceptible loquat (Eriobotrya japonica). Loquats replanted in an infection site that had been cleaned up by simply removing the diseased stump and visible residual roots remained healthy for a year. Taken together, P. noxius is not a soilborne pathogen, and diseased host tissues should be the focus of field sanitation and detection for BRR.

Two genes encoding GH10 xylanases are essential for the virulence of the oomycete plant pathogen Phytophthora parasitica.

Plant cell walls are pivotal battlegrounds between microbial pathogens and their hosts. To penetrate the cell wall and thereby to facilitate infection, microbial pathogens are equipped with a wide array of cell wall-degrading enzymes to depolymerize the polysaccharides in the cell wall. However, many of these enzymes and their role in the pathogenesis of microbial pathogens are not characterized, especially those from Oomycetes. In this study, we analyzed the function of four putative endo-beta-1,4-xylanase-encoding genes (ppxyn1-ppxyn4) from Phytophthora parasitica, an oomycete plant pathogen known to cause severe disease in a wide variety of plant species. All four genes belong to the glycoside hydrolase family 10 (GH10). Recombinant proteins of ppxyn1, ppxyn2, and ppxyn4 obtained from the yeast Pichia pastoris showed degrading activities toward birch wood xylan, but they behaved differently in terms of the conditions for optimal activity, thermostability, and durability. Quantitative RT-PCR revealed upregulated expression of all four genes, especially ppxyn1 and ppxyn2, during plant infection. In contrast, ppxyn3 was highly expressed in cysts and its close homolog, ppxyn4, in germinating cysts. To uncover the role of ppxyn1 and ppxyn2 in the pathogenesis of P. parasitica, we generated silencing transformants for these two genes by double-stranded RNA-mediated gene silencing. Silencing ppxyn1 and ppxyn2 reduced the virulence of P. parasitica toward tobacco (Nicotiana benthamiana) and tomato plants. These results demonstrate the crucial role of xylanase-encoding ppxyn1 and ppxyn2 in the infection process of P. parasitica.

Comparative and population genomic landscape of Phellinus noxius: A hypervariable fungus causing root rot in trees.

The order Hymenochaetales of white rot fungi contain some of the most aggressive wood decayers causing tree deaths around the world. Despite their ecological importance and the impact of diseases they cause, little is known about the evolution and transmission patterns of these pathogens. Here, we sequenced and undertook comparative genomic analyses of Hymenochaetales genomes using brown root rot fungus Phellinus noxius, wood-decomposing fungus Phellinus lamaensis, laminated root rot fungus Phellinus sulphurascens and trunk pathogen Porodaedalea pini. Many gene families of lignin-degrading enzymes were identified from these fungi, reflecting their ability as white rot fungi. Comparing against distant fungi highlighted the expansion of 1,3-beta-glucan synthases in P. noxius, which may account for its fast-growing attribute. We identified 13 linkage groups conserved within Agaricomycetes, suggesting the evolution of stable karyotypes. We determined that P. noxius has a bipolar heterothallic mating system, with unusual highly expanded ~60 kb A locus as a result of accumulating gene transposition. We investigated the population genomics of 60 P. noxius isolates across multiple islands of the Asia Pacific region. Whole-genome sequencing showed this multinucleate species contains abundant poly-allelic single nucleotide polymorphisms with atypical allele frequencies. Different patterns of intra-isolate polymorphism reflect mono-/heterokaryotic states which are both prevalent in nature. We have shown two genetically separated lineages with one spanning across many islands despite the geographical barriers. Both populations possess extraordinary genetic diversity and show contrasting evolutionary scenarios. These results provide a framework to further investigate the genetic basis underlying the fitness and virulence of white rot fungi.

PpMID1 Plays a Role in the Asexual Development and Virulence of Phytophthora parasitica.

Phytophthora parasitica is a notorious oomycete pathogen that causes severe disease in a wide variety of crop species. Infection of plants involves mainly its asexual life stage, including papillate sporangia and biflagellated zoospores, which are the primary dispersal and infection agents of this pathogen. Calcium signaling has been thought as the key regulator for sporangium formation and zoospore differentiation. However, not much is known about the molecular players involved in these processes. In Saccharomyces cerevisiae, mating pheromone-induced death 1 (MID1) encodes a component of a putative calcium channel. Here, we identified and characterized the function of PpMID1, an MID1 homolog from P. parasitica. The expression of PpMID1 was high in sporangia. Gene silencing of PpMID1 resulted in the formation of sporangia that lacked papilla and showed a tendency for direct germination. Notably, in response to cold shock to induce zoospore formation, these sporangia showed no sign of cytoplasmic cleavage and thereby failed to form zoospores. Nonetheless, the addition of CaCl2 or MgCl2 partially recovered the silenced sporangia phenotype, with the formation of papillate sporangia similar to those of the wild type and the release of zoospores upon cold shock. As well, virulence toward Nicotiana benthamiana was reduced in the PpMID1-silenced transformants. These results indicate a role of PpMID1 in the asexual development and virulence of P. parasitica.

Phellinus noxius: molecular diversity among isolates from Taiwan and its phylogenetic relationship with other species of Phellinus based on sequences of the ITS region.

BACKGROUND: Analysis of phylogenetic relationship of 91 isolates of Phellinus noxius obtained from 46 plant species in Taiwan did not show distinct grouping based on ITS sequences. RESULTS: However, the ITS nucleotides showed 20 different kinds of variations including single nucleotide polymorphisms, deletion and insertion in ITS1 and ITS2, but none in 5.8 S. The Taiwanese isolates of P. noxius were dividable into long (type L), median (type M) and short (type S) groups based on ITS sequence length. Two isolates with identical ITS sequence belonged to types L. Type M with 72 isolates was further divided into 33 subtypes, while types S with 17 isolates was further divided into two subtypes. CONCLUSION: Phylogenetic analysis of ITS sequences among Phellinus species showed that isolates of P. noxius were in the same clade distinctly separated from other Phellinus species.

Identification of the orsellinic acid synthase PKS63787 for the biosynthesis of antroquinonols in Antrodia cinnamomea.

Antrodia cinnamomea, an endemic basidiomycete used as a health food in Taiwan, is known to synthesize antroquinonols, which were reported to have notable medicinal potential in oncology and immunology. However, the biosynthetic pathway of these compounds is currently unclear. Our previous study showed that a pks63787 knockout mutant of A. cinnamomea (∆pks63787) is deficient in the biosynthesis of several aromatic metabolites. In this study, we pointed by phylogenetic analysis that pks63787 likely encodes an orsellinic acid synthase. Moreover, amendment of the cultural medium with orsellinic acid not only restores the ability of ∆pks63787 to produce its major pigment and other deficient metabolites, e.g., antroquinonols, but also enhances the productivity of several antroquinonols, including two new compounds 2 and 3. These results provide direct evidence that the PKS63787 is involved in the biosynthesis of antroquinonols and confirmed our hypothesis that the 6-methylcyclohexenone moiety was synthesized via the PKS63787-mediated polyketide pathway. In conclusion, PKS63787 might function as orsellinic acid synthase and orsellinic acid is an important precursor indispensable for the biosynthesis of the major pigment and antroquinonols in A. cinnamomea. To facilitate further basic or applied study, a putative biosynthesis pathway map of antroquinonols is proposed.

pks63787, a Polyketide Synthase Gene Responsible for the Biosynthesis of Benzenoids in the Medicinal Mushroom Antrodia cinnamomea.

Antrodia cinnamomea, a unique resupinate basidiomycete endemic to Taiwan, has potent medicinal activities. The reddish basidiocarps and mycelia generally exhibit abundant metabolites and higher biological activity. To investigate the pigments of A. cinnamomea, polyketide synthase (PKS) genes were characterized based on its partially deciphered genome and the construction of a fosmid library. Furthermore, a gene disruption platform was established via protoplast transformation and homologous recombination. Of four putative polyketide synthase genes, pks63787 was selected and disrupted in the monokaryotic wild-type (wt) strain f101. Transformant Δpks63787 was deficient in the synthesis of several aromatic metabolites, including five benzenoids and two benzoquinone derivatives. Based on these results, a biosynthetic pathway for benzenoid derivatives was proposed. The pks63787 deletion mutant not only displayed a reduced red phenotype compared to the wt strain but also displayed less 1,1-biphenyl-2-picrylhydrazyl free radical scavenging activity. This finding suggests that PKS63787 is responsible for the biosynthesis of pigments and metabolites related to the antioxidant activity of A. cinnamomea. The present study focuses on the functional characterization of the PKS gene, the fluctuations of its profile of secondary metabolites, and interpretation of the biosynthesis of benzenoids.

Development of oligonucleotide microarrays for simultaneous multi-species identification of Phellinus tree-pathogenic fungi.

Polyporoid Phellinus fungi are ubiquitously present in the environment and play an important role in shaping forest ecology. Several species of Phellinus are notorious pathogens that can affect a broad variety of tree species in forest, plantation, orchard and urban habitats; however, current detection methods are overly complex and lack the sensitivity required to identify these pathogens at the species level in a timely fashion for effective infestation control. Here, we describe eight oligonucleotide microarray platforms for the simultaneous and specific detection of 17 important Phellinus species, using probes generated from the internal transcribed spacer regions unique to each species. The sensitivity, robustness and efficiency of this Phellinus microarray system was subsequently confirmed against template DNA from two key Phellinus species, as well as field samples collected from tree roots, trunks and surrounding soil. This system can provide early, specific and convenient detection of Phellinus species for forestry, arboriculture and quarantine inspection, and could potentially help to mitigate the environmental and economic impact of Phellinus-related diseases.

The Genetic Structure of Phellinus noxius and Dissemination Pattern of Brown Root Rot Disease in Taiwan.

Since the 1990s, brown root rot caused by Phellinus noxius (Corner) Cunningham has become a major tree disease in Taiwan. This fungal pathogen can infect more than 200 hardwood and softwood tree species, causing gradual to fast decline of the trees. For effective control, we must determine how the pathogen is disseminated and how the new infection center of brown root rot is established. We performed Illumina sequencing and de novo assembly of a single basidiospore isolate Daxi42 and obtained a draft genome of ~40 Mb. By comparing the 12,217 simple sequence repeat (SSR) regions in Daxi42 with the low-coverage Illumina sequencing data for four additional P. noxius isolates, we identified 154 SSR regions with potential polymorphisms. A set of 13 polymorphic SSR markers were then developed and used to analyze 329 P. noxius isolates collected from 73 tree species from urban/agricultural areas in 14 cities/counties all around Taiwan from 1989 to 2012. The results revealed a high proportion (~98%) of distinct multilocus genotypes (MLGs) and that none of the 329 isolates were genome-wide homozygous, which supports a possible predominant outcrossing reproductive mode in P. noxius. The diverse MLGs exist as discrete patches, so brown root rot was most likely caused by multiple clones rather than a single predominant strain. The isolates collected from diseased trees near each other tend to have similar genotype(s), which indicates that P. noxius may spread to adjacent trees via root-to-root contact. Analyses based on Bayesian clustering, FST statistics, analysis of molecular variance, and isolation by distance all suggest a low degree of population differentiation and little to no barrier to gene flow throughout the P. noxius population in Taiwan. We discuss the involvement of basidiospore dispersal in disease dissemination.

Tomato SOBIR1/EVR Homologs Are Involved in Elicitin Perception and Plant Defense Against the Oomycete Pathogen Phytophthora parasitica.

During host-pathogen interactions, pattern recognition receptors form complexes with proteins, such as receptor-like kinases, to elicit pathogen-associated molecular pattern-triggered immunity (PTI), an evolutionarily conserved plant defense program. However, little is known about the components of the receptor complex, as are the molecular events leading to PTI induced by the oomycete Phytophthora pathogen. Here, we demonstrate that tomato (Solanum lycopersicum) SlSOBIR1 and SlSOBIR1-like genes are involved in defense responses to Phytophthora parasitica. Silencing of SlSOBIR1 and SlSOBIR1-like enhanced susceptibility to P. parasitica in tomato. Callose deposition, reactive oxygen species production, and PTI marker gene expression were compromised in SlSOBIR1- and SlSOBIR1-like-silenced plants. Interestingly, P. parasitica infection and elicitin (ParA1) treatment induced the relocalization of SlSOBIR1 from the plasma membrane to endosomal compartments and silencing of NbSOBIR1 compromised ParA1-mediated cell death on Nicotiana benthamiana. Moreover, the SlSOBIR1 kinase domain is indispensable for ParA1 to trigger SlSOBIR1 internalization and plant cell death. Taken together, these results support the idea of participation of solanaceous SOBIR1/EVR homologs in the perception of elicitins and indicate their important roles in plant basal defense against oomycete pathogens.

A novel elicitor protein from Phytophthora parasitica induces plant basal immunity and systemic acquired resistance.

The interaction between Phytophthora pathogens and host plants involves the exchange of complex molecular signals from both sides. Recent studies of Phytophthora have led to the identification of various apoplastic elicitors known to trigger plant immunity. Here, we provide evidence that the protein encoded by OPEL of Phytophthora parasitica is a novel elicitor. Homologues of OPEL were identified only in oomycetes, but not in fungi and other organisms. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed that OPEL is expressed throughout the development of P. parasitica and is especially highly induced after plant infection. Infiltration of OPEL recombinant protein from Escherichia coli into leaves of Nicotiana tabacum (cv. Samsun NN) resulted in cell death, callose deposition, the production of reactive oxygen species and induced expression of pathogen-associated molecular pattern (PAMP)-triggered immunity markers and salicylic acid-responsive defence genes. Moreover, the infiltration conferred systemic resistance against a broad spectrum of pathogens, including Tobacco mosaic virus, the bacteria wilt pathogen Ralstonia solanacearum and P. parasitica. In addition to the signal peptide, OPEL contains three conserved domains: a thaumatin-like domain, a glycine-rich protein domain and a glycosyl hydrolase (GH) domain. Intriguingly, mutation of a putative laminarinase active site motif in the predicted GH domain abolished its elicitor activity, which suggests enzymatic activity of OPEL in triggering the defence response.

Generation and analysis of the expressed sequence tags from the mycelium of Ganoderma lucidum.

Ganoderma lucidum (G. lucidum) is a medicinal mushroom renowned in East Asia for its potential biological effects. To enable a systematic exploration of the genes associated with the various phenotypes of the fungus, the genome consortium of G. lucidum has carried out an expressed sequence tag (EST) sequencing project. Using a Sanger sequencing based approach, 47,285 ESTs were obtained from in vitro cultures of G. lucidum mycelium of various durations. These ESTs were further clustered and merged into 7,774 non-redundant expressed loci. The features of these expressed contigs were explored in terms of over-representation, alternative splicing, and natural antisense transcripts. Our results provide an invaluable information resource for exploring the G. lucidum transcriptome and its regulation. Many cases of the genes over-represented in fast-growing dikaryotic mycelium are closely related to growth, such as cell wall and bioactive compound synthesis. In addition, the EST-genome alignments containing putative cassette exons and retained introns were manually curated and then used to make inferences about the predominating splice-site recognition mechanism of G. lucidum. Moreover, a number of putative antisense transcripts have been pinpointed, from which we noticed that two cases are likely to reveal hitherto undiscovered biological pathways. To allow users to access the data and the initial analysis of the results of this project, a dedicated web site has been created at http://csb2.ym.edu.tw/est/.

Functional characterization of a gene family encoding Polygalacturonases in Phytophthora parasitica.

Phytophthora parasitica is an oomycete plant pathogen that causes severe disease in a wide variety of plant species. In our previous study, we discovered a multigene family encoding endopolygalacturonases (endoPG) in Phytophthora parasitica. Here, we screened the genomic library of Phytophthora parasitica for the genes encoding endoPG named pppg2 through pppg10, and analyzed their functions. Results obtained by real-time quantitative reverse transcriptase-polymerase chain reaction demonstrated that some of these genes are highly induced during plant infection, which suggests their important roles in the pathogenesis of Phytophthora parasitica. Analysis by in-gel activity assay of recombinant proteins obtained from Pichia pastoris indicated that each of these genes encodes a functional endoPG. Investigation of the function of pppg genes in planta by a Potato virus X agroinfection system in tobacco revealed that each pppg caused specific effects, varying from no symptoms to dwarfism, necrosis, leaf curl, silvery leaf, and cracks in leaf stalks. Appearance of these effects depends on the expression of a pppg protein with a normal active site in the apoplast. These results indicated that each pppg plays a distinct role in the decomposition of plant cell wall.

Selection of internal control genes for real-time quantitative RT-PCR assays in the oomycete plant pathogen Phytophthora parasitica.

Real-time quantitative reverse transcription-PCR (qRT-PCR) has become one of the most commonly used methods for RNA quantification in recent years. To obtain reliable results with biological significance, it is important that qRT-PCR data are normalized with a proper internal control. In this study, 18 housekeeping genes were selected and evaluated for their potential as a suitable internal control for study of gene expression in the oomycete plant pathogen Phytophthora parasitica. Analysis of qRT-PCR data using the geNorm software indicated that, although commonly used as internal controls, beta-actin (ACT) and translation elongation factor 1alpha (eEF1A) might not be the best choice due to variable expression across different life stages of P. parasitica. Instead, other genes would serve as better controls, including ubiquitin-conjugating enzyme (Ubc), WS21, and beta-tubulin (Tub-b) for 'asexual stage,' Ubc and Tub-b for 'sexual reproduction,' while Ubc and WS21 for the stage of pathogenesis, because of their constant expression levels in each given subset of RNA samples. Although normalization with more than one gene would generate more reliable results, use of a single stably expressed gene as an internal control would suffice for accurate data normalization in some experiments.

Cloning and analysis of pppg1, an inducible endopolygalacturonase gene from the oomycete plant pathogen Phytophthora parasitica.

Phytophthora parasitica is an oomycete plant pathogen that causes severe disease in a wide variety of crops. Here, we report the isolation of a gene, named pppg1, which encodes an extracellular endopolygalacturonase in P. parasitica. Both cDNA and a genomic clone were isolated and sequenced. The pppg1 gene showed standard characteristics with respect to core promoter and intron sequences of Phytophthora. The predicted protein of pppg1 has a calculated molecular mass of 39.7 kDa and a pI value of 5.2, and contains a putative signal peptide of 20 amino acid residues on the N-terminus. The deduced amino acid sequence is highly conserved with those of other Phytophthora and fungal endopolygalacturonases. Analysis by reverse transcription followed by real-time quantitative polymerase chain reaction showed that transcription of pppg1 was repressed by glucose, but induced by pectin in the culture. Moreover, pppg1 is highly expressed during interaction of P. parasitica with the host plant, suggesting its involvement in the process of host infection. Heterologous expression of pppg1 in Pichia pastoris produced proteins with molecular mass ranging from 75 to 200 kDa, very likely due to differential glycosylation by the yeast. Deglycosylation of the recombinant protein resulted in a complete loss of the endopolygalacturonase activity.

The period gene of the German cockroach and its novel linking power between vertebrate and invertebrate.

Circadian clock protein PERIOD (PER) is essential for the endogenous clockworks in diverse lineages within Metazoa, but the protein sequences, the clock protein interactions, and the photic responses are variant and different between vertebrate and invertebrate PER homologs. Here we identified the German cockroach PER homologs and found it could bridge the huge phylogenetic gap and make possible a more precise protein sequence comparison between vertebrate and invertebrate PER homologs. Seven blocks of conserved regions (c1-c7) interspersed within PER proteins were defined, and two new significant homologies were found in the upstream portion of c3 region and in the c7 region, respectively. In addition, we found all dipteran insects PER homologs lack the c7 region and its following amino acid residues. Our results not only reveal the homology and divergence, but also imply the constraint and plasticity of divergent PER proteins during the course of evolution. These findings lay a solid foundation for understanding the general and divergent properties of circadian clockworks in diverse lineages within Metazoa.