Five Reasons to Consider Phytophthora infestans a Reemerging Pathogen.

Phytophthora infestans has been a named pathogen for well over 150 years and yet it continues to "emerge", with thousands of articles published each year on it and the late blight disease that it causes. This review explores five attributes of this oomycete pathogen that maintain this constant attention. First, the historical tragedy associated with this disease (Irish potato famine) causes many people to be fascinated with the pathogen. Current technology now enables investigators to answer some questions of historical significance. Second, the devastation caused by the pathogen continues to appear in surprising new locations or with surprising new intensity. Third, populations of P. infestans worldwide are in flux, with changes that have major implications to disease management. Fourth, the genomics revolution has enabled investigators to make tremendous progress in terms of understanding the molecular biology (especially the pathogenicity) of P. infestans. Fifth, there remain many compelling unanswered questions.

Highlights of the mini-symposium on extracellular vesicles in inter-organismal communication, held in Munich, Germany, August 2018.

All living organisms secrete molecules for intercellular communication. Recent research has revealed that extracellular vesicles (EVs) play an important role in inter-organismal cell-to-cell communication by transporting diverse messenger molecules, including RNA, DNA, lipids and proteins. These discoveries have raised fundamental questions regarding EV biology. How are EVs biosynthesized and loaded with messenger/cargo molecules? How are EVs secreted into the extracellular matrix? What are the EV uptake mechanisms of recipient cells? As EVs are produced by all kind of organisms, from unicellular bacteria and protists, filamentous fungi and oomycetes, to complex multicellular life forms such as plants and animals, basic research in diverse model systems is urgently needed to shed light on the multifaceted biology of EVs and their role in inter-organismal communications. To help catalyse progress in this emerging field, a mini-symposium was held in Munich, Germany in August 2018. This report highlights recent progress and major questions being pursued across a very diverse group of model systems, all united by the question of how EVs contribute to inter-organismal communication.

Novel quorum-sensing-controlled genes in Erwinia carotovora subsp. carotovora: identification of a fungal elicitor homologue in a soft-rotting bacterium.

Seven new genes controlled by the quorum-sensing signal molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) have been identified in Erwinia carotovora subsp. carotovora. Using TnphoA as a mutagen, we enriched for mutants defective in proteins that could play a role in the interaction between E. carotovora subsp. carotovora and its plant hosts, and identified NipEcc and its counterpart in E. carotovora subsp. atroseptica. These are members of a growing family of proteins related to Nep1 from Fusarium oxysporum which can induce necrotic responses in a variety of dicotyledonous plants. NipEcc produced necrosis in tobacco, NipEca affected potato stem rot, and both affected virulence in potato tubers. In E. carotovora subsp. carotovora, nip was shown to be subject to weak repression by the LuxR family regulator, EccR, and may be regulated by the negative global regulator RsmA.

A cysteine protease gene is expressed early in resistant potato interactions with Phytophthora infestans.

A potato cysteine protease (cyp) cDNA expressed at an early stage of an incompatible interaction with Phytophthora infestans was isolated. Both the nucleotide and deduced amino acid sequences are highly homologous to those of a tomato cysteine protease, CYP1. Striking protein similarity to all known cathepsins in animals, particularly cathepsin K, was also observed. However, unlike cathepsins, a granulin binding domain is located near the carboxyl terminus of the putative CYP protein. In animals, granulins bind to receptors in the plasma membrane and signal cell growth and division. A ribonuclease protection assay demonstrated that the cyp gene is tightly regulated and is induced 15 h post inoculation with P. infestans in potato leaves either with high field resistance or in which a resistance (R) gene is activated. We conclude that a common signaling pathway is activated in each form of resistance.

A potato gene encoding a WRKY-like transcription factor is induced in interactions with Erwinia carotovora subsp. atroseptica and Phytophthora infestans and is coregulated with class I endochitinase expression.

A potato gene encoding a putative WRKY protein was isolated from a cDNA library enriched by suppression subtractive hybridization for sequences upregulated 1 h postinoculation with Erwinia carotovora subsp. atroseptica. The cDNA encodes a putative polypeptide of 172 amino acids, containing a single WRKY domain with a zinc finger motif and preceded by a potential nuclear localization site. St-WRKY1 was strongly upregulated in compatible, but only weakly in incompatible, interactions with Phytophthora infestans where, in all cases, it was coregulated with class I endochitinase, associating its expression with a known defense response. Whereas St-WRKY1 was strongly induced by E. carotovora culture filtrate (CF), confirming it to be an elicitor-induced gene, no such induction was detected after treatment with salicylic acid, methyl jasmonate, ethylene, or wounding. St-WRKY1 was upregulated by treatment of potato leaves with CFs from recombinant Escherichia coli containing plasmids expressing E. carotovora pectate lyase genes pelB and pelD, suggesting that either proteins encoded by these genes, or oligogalacturonides generated by their activity, elicit a potato defense pathway associated with St-WRKY1.

Nucleotide sequence of a gene from Phanerochaete chrysosporium that shows homology to the facA gene of Aspergillus nidulans.

Heterologous hybridisation was used to isolate a genomic DNA sequence from Phanerochaete chrysosporium using the facA (acetyl CoA synthetase) gene from Aspergillus nidulans as a probe. The cloned sequence hybridises to a 2.2 kb transcript in poly(A)+ RNA prepared from mycelium grown on acetate as the sole carbon source. Comparison of the DNA sequence obtained with those of the A. nidulans facA and N. crassa acu5 genes reveals an ORF that appears to be interrupted by five typical fungal introns. Two possible candidates for the translation initiation codon were observed. Homology with the facA and acu5 genes is revealed after the second ATG codon.

Targeted differential display of abundantly expressed sequences from the basidiomycete Phanerochaete chrysosporium which contain regions coding for fungal cellulose-binding domains.

Cellulose-binding domains (CBDs) are present in the majority of fungal cellulases studied to-date. This work describes the use of targeted differential display, employing degenerate primers designed to anneal to variants of a region conserved in fungal CBDs, each in combination with an oligo-dT primer, to PCR-amplify cDNA sequences containing regions coding for such domains from Phanerochaete chrysosporium. After growth on either Avicel or carboxymethyl cellulose (CMC), five distinct, abundantly expressed cDNA sequences were obtained. Two of these originated from transcripts of the previously characterised cbhI.1 and cbhI.2 genes, whereas three were from novel genes. One of the latter was isolated only after growth on CMC. No such sequences were obtained after growth on xylan, suggesting that the expression of sequences containing such regions is down-regulated on this substrate. The use of targeted differential display both for isolating novel sequences and for studying the expression of known genes within a family is discussed.

Phytophthora infestans enters the genomics era.

UNLABELLED: summary Phytophthora infestans, cause of late-blight, is the most devastating disease of potato world-wide. Recent years have seen a dramatic intensification in molecular biological studies of P. infestans, including the development of novel tools for transformation and gene silencing and the resources for genetical, transcriptional and physical mapping of the genome. This review will focus on the increasing efforts to use these resources to discover the genetic bases of pathogenicity, avirulence and host-specificity. TAXONOMY: Phytophthora infestans (Mont.) de Bary-Kingdom Chromista, Phylum Oomycota, Order Peronosporales, Family Peronosporaceae, Genus Phytophthora, of which it is the type species. HOST RANGE: Infects a wide range of solanaceous species. Economically important hosts are potato, tomato, eggplant and some other South American hosts (tree tomato and pear melon) on which it causes late blight. Disease symptoms: Infected foliage is initially yellow, becomes water soaked and eventually blackens. Leaf symptoms comprise purple-black or brown-black lesions at the leaf tip, later spreading across the leaf to the stem. Whitish masses of sporangia develop on the underside of the leaf. Tubers become infected later in the season and, in the early stages, consist of slightly brown or purple blotches on the skin. In damp soils the tuber decays rapidly before harvest. Tuber infection is quickly followed by secondary fungal or bacterial infection known as 'wet rot'. Useful web sites:http://www.ncgr.org/pgc/; http://www.oardc.ohio-state.edu/phytophthora/.

Substrate-dependent differential splicing of introns in the regions encoding the cellulose binding domains of two exocellobiohydrolase I-like genes in Phanerochaete chrysosporium.

Recently, we have shown differential splicing of an intron in the cbhI.2 gene of Phanerochaete chrysosporium ME446; this intron lies within the region of the gene encoding the cellulose binding domain (P.F.G. Sims, M. S. Soares-Felipe, Q. Wang, M.E. Gent, C. Tempelaars, and P. Broda, Mol. Microbiol. 12:209-216, 1994). Here, we show that such differential splicing occurs in the cbhI.1 gene of this fungus as well as in the cbhI.2 gene and that this phenomenon is substrate dependent. Avicel elicits the synthesis of both classes of mRNA transcripts from both of these genes. In contrast, carboxymethyl cellulose predominantly elicits the synthesis of fully spliced transcripts from both genes. Such differential splicing might allow this fungus to regulate the specificities of substrate binding for these cellulases.

A reporter system for analysis of regulatable promoter functions in the basidiomycete fungus Phanerochaete chrysosporium.

To establish a system for functional analysis of regulatable gene promoters involved in lignocellulose degradation by Phanerochaete chrysosporium, a DNA construct was made in which a minimal promoter region, 410 bp of the 5' untranslated region (UTR) of the cbhI.1 gene of P. chrysosporium, was fused to the phlR sequence of Streptoallotiechus hindustanus. This construct was used to transform P. chrysosporium to phleomycin resistance. Southern blot analysis revealed that the incoming DNA was maintained extrachromosomally in the transformants. The donor DNA was methylated by the fungus; inhibition of such methylation allowed transformants to be distinguished from 'false-positive' phlR colonies. Reverse transcriptase-polymerase chain reaction analysis of gene expression revealed that the cbhI.1 5' UTR/phlR sequence construct was transcribed, but that an intron within the cbhI.1 promoter was not excised from transcripts of the transforming DNA construct. Moreover, catabolite repression of cbhI.1 expression is relaxed in the construct. These observations suggest that normal function of the cbhI.1 promoter requires more than 410 bp.

Isolation, characterization, and analysis of the expression of the cbhII gene of Phanerochaete chrysosporium.

Two cDNA sequences representing putative allelic variants of the Phanerochaete chrysosporium cbhII gene were isolated by hybridization to the Trichoderma reesei cbhII gene. Both of the equivalent genomic sequences were subsequently isolated by the inverse PCR technique. DNA sequencing showed that the cbhII open reading frame of 1,380 bp codes for a putative polypeptide of 460 amino acids which is interrupted by six introns. The domain structure found in T. reesei cbhII is conserved in the equivalent P. chrysosporium protein. The overall similarity between the two gene products is 54%, with the region of highest conservation being found in the cellulose-binding domain (65%). Unlike the cbhI gene of P. chrysosporium, cbhII does not appear to be a member of a class of closely related genes. CBHII is a new member of family B of the beta-1, 4-glucanases. Alignment of the P. chrysosporium and T. reesei CBHII protein sequences showed that all of the residues important for the formation of the extended loops of the catalytic domain and those residues that are involved in the catalytic action of the T. reesei enzyme are also present in the P. chrysosporium equivalent. The profiles of cbh gene expression in P. chrysosporium reveal that while cbhI.1 and cbhI.2 could be coregulated, cbhII can be independently controlled. The latter is so far the only cellulase gene found to be expressed when the fungus is grown on oat spelt arabinoxylan, suggesting that it may play an active role in the xylanolytic as well as the cellulolytic systems.

A competitive PCR-based method for the detection and quantification of Erwinia carotovora subsp. atroseptica On potato tubers.

A PCR-based method was developed for the simultaneous detection and quantification of the potato pathogen Erwinia carotovora subsp. atroseptica (Eca) on potato tubers. The method incorporates a competitor PCR template cloned into Escherichia coli in vector pGEM-T (E. coli 4R l/l). Predetermined numbers of E. coli 4R were added to potato peel extract, either pre-inoculated with Eca or from naturally contaminated tubers, and Eca numbers estimated by comparing the ratio of products generated from Eca target DNA and competitor template DNA following PCR. Estimates of Eca numbers were consistent with counts obtained on crystal violet pectate medium and immunofluorescence colony staining. Unlike these methods, however, the PCR-based method is not affected by the presence of other erwinias and saprophytes and is able to detect all serogroups of Eca. Based on this method, a key was produced relating product ratios, obtained following PCR from contaminated tuber stocks, to the likelihood of blackleg disease incidence. This is the first quantitative PCR-based detection method described for Eca and is the first for any bacterial plant pathogen to incorporate a DNA extraction control.

PCR-mediated analysis of lignocellulolytic gene transcription by Phanerochaete chrysosporium: substrate-dependent differential expression within gene families.

We compare the kinetics of appearance of supernatant enzyme activities (lignin peroxidase, manganese peroxidase, and cellulase) and gene expression (LIG, mnp, and cbhI gene families and the unique cbhII gene) in Phanerochaete chrysosporium ME446 when grown on four different carbon sources: ball-milled straw, representing the natural substrate lignocellulose; Avicel as a crystalline cellulose; and high and low concentrations of glucose, in all cases with limiting nitrogen. PCR-based technology utilizing pairs of primers specific for particular genes showed that there is differential expression between and within the families. There were a number of instances of mRNA species being present only on a single day, implying tight regulation of lignocellulose degradation at the mRNA level. The patterns of extracellular enzyme activities and mnp and cbh gene expression are similar whereas LIG gene expression can be detected when no corresponding enzyme activity is observed in the extracellular supernatant. The enzyme produced under these conditions is presumably sequestered by the mycelium and is likely to be functionally significant. Another striking result is that cellulose, in the form of Avicel, elicits the expression of three LIG gene for which there is no expression under the same conditions with the other carbon sources.

Lignocellulose degradation by Phanerochaete chrysosporium: gene families and gene expression for a complex process.

Phanerochaete chrysosporium completely degrades lignocellulose. The most recalcitrant component, lignin, is oxidized by the radical products of lignin and manganese peroxidases, whereas cellulose and hemicellulose are hydrolysed. Both peroxidases and cellulases exist as complex families at both the DNA and protein levels. The lignin peroxidases may function principally when mycelium-bound and, therefore, undetectable in culture supernatants. Moreover, methods for the study of P. chrysosporium must be applicable to solid substrate as well as liquid-culture conditions. For these reasons, detailed studies of gene expression, made possible by the reverse transcriptase-polymerase chain reaction method, are essential. Such studies reveal that gene families are subject to differential expression. The cellulase system has some differences from that of Trichoderma reesei; the distinction made between the activities of exocellobiohydrolases and endoglucanases needs to be re-appraised in both species. Current studies also seek to reconstruct the systems of degradation of lignocellulose and its individual components by heterologous expression of individual proteins in recombinant systems, and their use in mechanistic studies singly and in combinations.

Physical mapping across an avirulence locus of Phytophthora infestans using a highly representative, large-insert bacterial artificial chromosome library.

The oomycete plant pathogen Phytophthora infestans is the causal agent of late blight, one of the most devastating diseases of potato worldwide. As part of efforts to clone avirulence (Avr) genes and pathogenicity factors from P. infestans, we have constructed a bacterial artificial chromosome (BAC) library from an isolate containing six Avr genes. The BAC library comprises clones with an average insert size of 98 kb and represents an estimated 10 genome equivalents. A three-dimensional pooling strategy was developed to screen the BAC library for amplified fragment length polymorphism (AFLP) markers, as this type of marker has been extensively used in construction of a P. infestans genetic map. Multiple positive clones were identified for each AFLP marker tested. The pools were used to construct a contig of 11 BAC clones in a region of the P. infestans genome containing a cluster of three avirulence genes. The BAC contig is predicted to encompass the Avr11 locus but mapping of the BAC ends will be required to determine if the Avr3 and Avr10 loci are also present in the BAC contig. These results are an important step towards the positional cloning of avirulence genes from P. infestans, and the BAC library represents a valuable resource for largescale studies of oomycete genome organisation and gene content.

Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors.

The bacterial family Enterobacteriaceae is notable for its well studied human pathogens, including Salmonella, Yersinia, Shigella, and Escherichia spp. However, it also contains several plant pathogens. We report the genome sequence of a plant pathogenic enterobacterium, Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043, the causative agent of soft rot and blackleg potato diseases. Approximately 33% of Eca genes are not shared with sequenced enterobacterial human pathogens, including some predicted to facilitate unexpected metabolic traits, such as nitrogen fixation and opine catabolism. This proportion of genes also contains an overrepresentation of pathogenicity determinants, including possible horizontally acquired gene clusters for putative type IV secretion and polyketide phytotoxin synthesis. To investigate whether these gene clusters play a role in the disease process, an arrayed set of insertional mutants was generated, and mutations were identified. Plant bioassays showed that these mutants were significantly reduced in virulence, demonstrating both the presence of novel pathogenicity determinants in Eca, and the impact of functional genomics in expanding our understanding of phytopathogenicity in the Enterobacteriaceae.