The role of strigolactones and ethylene in disease caused by Pythium irregulare

Plant hormones play key roles in defence against pathogen attack. Recent work has begun to extend this role to encompass not just the traditional disease/stress hormones, such as ethylene, but also growth‐promoting hormones. Strigolactones (SLs) are the most recently defined group of plant hormones with important roles in plant–microbe interactions, as well as aspects of plant growth and development, although the knowledge of their role in plant–pathogen interactions is extremely limited. The oomycete Pythium irregulare is a poorly controlled pathogen of many crops. Previous work has indicated an important role for ethylene in defence against this oomycete. We examined the role of ethylene and SLs in response to this pathogen in pea (Pisum sativum L.) at the molecular and whole‐plant levels using a set of well‐characterized hormone mutants, including an ethylene‐insensitive ein2 mutant and SL‐deficient and insensitive mutants. We identified a key role for ethylene signalling in specific cell types that reduces pathogen invasion, extending the work carried out in other species. However, we found no evidence that SL biosynthesis or response influences the interaction of pea with P. irregulare or that synthetic SL influences the growth or hyphal branching of the oomycete in vitro. Future work should seek to extend our understanding of the role of SLs in other plant interactions, including with other fungal, bacterial and viral pathogens, nematodes and insect pests.     

Site‐specific distribution of oak rhizosphere‐associated oomycetes revealed by cytochrome c oxidase subunit II metabarcoding

The phylum Oomycota comprises important tree pathogens like Phytophthora quercina, involved in central European oak decline, and Phytophthora cinnamomi shown to affect holm oaks among many other hosts. Despite the importance to study the distribution, dispersal and niche partitioning of this phylum, metabarcoding surveys, and studies considering environmental factors that could explain oomycete community patterns are still rare. We investigated oomycetes in the rhizosphere of evergreen oaks in a Spanish oak woodland using metabarcoding based on Illumina sequencing of the taxonomic marker cytochrome c oxidase subunit II (cox2). We developed an approach amplifying a 333 bp long fragment using the forward primer Hud‐F (Mycologia, 2000) and a reverse primer found using DegePrime (Applied and Environmental Microbiology, 2014). Factors reflecting topo‐edaphic conditions and tree health were linked to oomycete community patterns. The majority of detected OTUs belonged to the Peronosporales. Most taxa were relatives of the Pythiaceae, but relatives of the Peronosporaceae and members of the Saprolegniales were also found. The most abundant OTUs were related to Globisporangium irregulare and P. cinnamomi, both displaying strong site‐specific patterns. Oomycete communities were strongly correlated with the environmental factors: altitude, crown foliation, slope and soil skeleton and soil nitrogen. Our findings illustrate the significance of small scale variation in habitat conditions for the distribution of oomycetes and highlight the importance to study oomycete communities in relation to such ecological patterns.     

Characterization of microsatellite markers in the interspecific hybrid Phytophthora alni ssp. alni,and cross‐amplification with related taxa

Phytophthora alni ssp. alni is an interspecific hybrid oomycete causing a large‐scale decay of alders throughout Europe. In this study we developed a set of 10 microsatellite markers that shows promise for population studies and for studying hybridization events between the parental species of the hybrid. Moreover, the genotype and the ploidy of the different subspecies of P. alni might be inferred from the quantitative ratio of amplified genome‐specific alleles. Nine primer pairs cross amplified with the related species Phytophthora cambivora and Phytophthora fragariae and yielded distinct alleles.     

Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9

Phytophthora sojae is an oomycete pathogen of soybean. As a result of its economic importance, P. sojae has become a model for the study of oomycete genetics, physiology and pathology. The lack of efficient techniques for targeted mutagenesis and gene replacement have long hampered genetic studies of pathogenicity in Phytophthora species. Here, we describe a CRISPR/Cas9 system enabling rapid and efficient genome editing in P. sojae. Using the RXLR effector gene Avr4/6 as a target, we observed that, in the absence of a homologous template, the repair of Cas9‐induced DNA double‐strand breaks (DSBs) in P. sojae was mediated by non‐homologous end‐joining (NHEJ), primarily resulting in short indels. Most mutants were homozygous, presumably as a result of gene conversion triggered by Cas9‐mediated cleavage of non‐mutant alleles. When donor DNA was present, homology‐directed repair (HDR) was observed, which resulted in the replacement of Avr4/6 with the NPT II gene. By testing the specific virulence of several NHEJ mutants and HDR‐mediated gene replacements in soybean, we have validated the contribution of Avr4/6 to recognition by soybean R gene loci, Rps4 and Rps6, but also uncovered additional contributions to resistance by these two loci. Our results establish a powerful tool for the study of functional genomics in Phytophthora, which provides new avenues for better control of this pathogen.     

The Impact of Fungal Extracts on Leaf Litter on the Food Preference of Gammarus roeselii

We investigated the effect of methanol and methanol/methylene chloride extracts of the oomycete Pythium sp. JN 1‐b and of the fungi Ascomycete sp. PVSo8, Fusarium sporotrichoides, and Cylindrocarpon sp. 94‐2057 on the food preference of Gammarus roeselii. The preference for leaf discs coated with these extracts compared to uncoated leaf discs was tested in food‐choice assays. Methanol extracts of all strains repelled G. roeselii, and the effect of the extract concentration on relative consumption was strain specific. The repellent effect of these extracts, especially of extracts of Cylindrocarpon sp., decreased when the fungi were grown on leaf extract medium as opposed to synthetic medium containing sucrose. None of the methanol/methylene chloride extracts affected the food preference of the gammarid. We conclude that biologically active compounds were extracted from fungi and an oomycete were soluble in methanol but not in methanol/methylene chloride. Only repellent activity was observed with the extracts, and relative ratios of repellents and attractants might determine the consumption of fungi by G. roeselii. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Genetic toolkits of the red alga Pyropia tenera against the three most common diseases in Pyropia farms

Disease outbreaks devastate Pyropia aquaculture farms every year. The three most common and serious diseases are Olpidiopsis‐blight and red‐rot disease caused by oomycete pathogens and green‐spot disease caused by the PyroV1 virus. We hypothesized that a basic genetic profile of molecular defenses will be revealed by comparing and analyzing the genetic response of Pyropia tenera against the above three pathogens. RNAs isolated from infected thalli were hybridized onto an oligochip containing 15,115 primers designed from P. tenera expressed sequence tags (EST)s. Microarray profiles of the three diseases were compared and interpreted together with histochemical observation. Massive amounts of reactive oxygen species accumulated in P. tenera cells exposed to oomycete pathogens. Heat shock genes and serine proteases were the most highly up‐regulated genes in all infection experiments. Genes involved in RNA metabolism, ribosomal proteins and antioxidant metabolism were also highly up‐regulated. Genetic profiles of P. tenera in response to pathogens were most similar between the two biotrophic pathogens, Olpidiopsis pyropiae and PyroV1 virus. A group of plant resistance genes were specifically regulated against each pathogen. Our results suggested that disease response in P. tenera consists of a general constitutive defense and a genetic toolkit against specific pathogens.     

Soil pathogen communities associated with native and non‐native Phragmites australis populations in freshwater wetlands

Soil pathogens are believed to be major contributors to negative plant–soil feedbacks that regulate plant community dynamics and plant invasions. While the theoretical basis for pathogen regulation of plant communities is well established within the plant–soil feedback framework, direct experimental evidence for pathogen community responses to plants has been limited, often relying largely on indirect evidence based on above‐ground plant responses. As a result, specific soil pathogen responses accompanying above‐ground plant community dynamics are largely unknown. Here, we examine the oomycete pathogens in soils conditioned by established populations of native noninvasive and non‐native invasive haplotypes of Phragmites australis (European common reed). Our aim was to assess whether populations of invasive plants harbor unique communities of pathogens that differ from those associated with noninvasive populations and whether the distribution of taxa within these communities may help to explain invasive success. We compared the composition and abundance of pathogenic and saprobic oomycete species over a 2‐year period. Despite a diversity of oomycete taxa detected in soils from both native and non‐native populations, pathogen communities from both invaded and noninvaded soils were dominated by species of Pythium. Pathogen species that contributed the most to the differences observed between invaded and noninvaded soils were distributed between invaded and noninvaded soils. However, the specific taxa in invaded soils responsible for community differences were distinct from those in noninvaded soils that contributed to community differences. Our results indicate that, despite the phylogenetic relatedness of native and non‐native P. australis haplotypes, pathogen communities associated with the dominant non‐native haplotype are distinct from those of the rare native haplotype. Pathogen taxa that dominate either noninvaded or invaded soils suggest different potential mechanisms of invasion facilitation. These findings are consistent with the hypothesis that non‐native plant species that dominate landscapes may “cultivate” a different soil pathogen community to their rhizosphere than those of rarer native species.     

Probing formation of cargo/importin‐α transport complexes in plant cells using a pathogen effector

Importin‐αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin‐α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin‐α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin‐α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co‐opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin‐α paralogs from Arabidopsis thaliana. A crystal structure of the importin‐α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin‐αs expressed in rosette leaves have an almost identical NLS‐binding site. Comparison of the importin‐α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin‐α, sequence variation at the importin‐α NLS‐binding sites and tissue‐specific expression levels of importin‐αs determine formation of cargo/importin‐α transport complexes in plant cells.     

Obligate biotrophic pathogens of the genus Albugo are widespread as asymptomatic endophytes in natural populations of Brassicaceae

Mutualistic interactions of plants with true fungi are a well‐known and widespread phenomenon, which includes mycorrhiza and non‐mycorrhizal endophytes like species of Epichloë. Despite the fact that these organisms intrude into plants, neither strong defence reactions nor the onset of symptoms of disease can be observed in most or even all infested plants, in contrast to endophytic pathogens. Oomycetes are fungal‐like organisms belonging to the kingdom Straminipila, which includes diatoms and seaweeds. Although having evolved many convergent traits with true fungi and occupying similar evolutionary niches, widespread oomycete endophytes are not known to date, although more than 500 endophytic pathogens, including species of the obligate biotrophic genus Albugo, have been described. Here, we report that oomycetes of the genus Albugo are widespread in siliques of natural host populations. A total of 759 plants, encompassing four genera with rare reports of white blister incidents and one with common incidents, were collected from 25 sites in Germany. Nested PCR with species‐specific primers revealed that 5–27% of the hosts with rare disease incidence carried asymptomatic Albugo in their siliques, although only on a single plant of 583 individuals, an isolated pustule on a single leaf could be observed. Control experiments confirmed that these results were not because of attached spores, but because of endophytic mycelium. Vertical inheritance of oomycete infections has been reported for several plant pathogens, and it seems likely that in nature this way of transmission plays an important role in the persistence of asymptomatic endophytic Albugo species.     

“Ectrogella” Parasitoids of the Diatom Licmophora sp. are Polyphyletic

The diatom genera Licmophora and Fragilaria are frequent epiphytes on marine macroalgae and can be infected by intracellular parasitoids traditionally assigned to the oomycete genus Ectrogella. Much debate and uncertainty remains about the taxonomy of these oomycetes, not least due to their morphological and developmental plasticity. Here, we used single‐cell techniques to obtain partial sequences of the parasitoids 18S and cox2 genes. The former falls into two recently identified clades of Pseudo‐nitzschia parasites temporarily named OOM_1_2 and OOM_2, closely related to the genera of brown and red algal pathogens Anisolpidium and Olpidiopsis. A third group of sequences falls at the base of the red algal parasites assigned to Olpidiopsis. In one instance, two oomycete parasitoids seemed to co‐exist in a single diatom cell; this co‐occurrence of distinct parasitoid taxa not only within a population of diatom epiphytes, but also within the same host cell, possibly explains the ongoing confusion in the taxonomy of these parasitoids. We demonstrate the polyphyly of Licmophora parasitoids previously assigned to Ectrogella (sensu Sparrow, 1960) and show that parasites of red algae assigned to the genus Olpidiopsis are most likely not monophyletic. We conclude that combining single‐cell microscopy and molecular methods is necessary for their full characterisation.     

A PCR assay for the quantification of growth of the oomycete pathogen Hyaloperonospora arabidopsidis in Arabidopsis thaliana

The accurate quantification of disease severity is important for the assessment of host–pathogen interactions in laboratory or field settings. The interaction between Arabidopsis thaliana and its naturally occurring downy mildew pathogen, Hyaloperonospora arabidopsidis (Hpa), is a widely used reference pathosystem for plant–oomycete interactions. Current methods for the assessment of disease severity in the Arabidopsis–Hpa interaction rely on measurements at the terminal stage of pathogen development; namely, visual counts of spore‐producing structures or the quantification of spore production with a haemocytometer. These assays are useful, but do not offer sensitivity for the robust quantification of small changes in virulence or the accurate quantification of pathogen growth prior to the reproductive stage. Here, we describe a quantitative real‐time polymerase chain reaction (qPCR) assay for the monitoring of Hpa growth in planta. The protocol is rapid, inexpensive and can robustly distinguish small changes in virulence. We used this assay to investigate the dynamics of early Hpa mycelial growth and to demonstrate the proof of concept that this assay could be used in screens for novel oomycete growth inhibitors.     

Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol‐3‐phosphate‐binding proteins

The internalization of some oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors' cell entry receptor, phosphatidylinositol‐3‐phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants to secrete PI3P‐binding proteins. In this study, we tested this strategy using the chocolate tree Theobroma cacao. Transient expression and secretion of four different PI3P‐binding proteins in detached leaves of T. cacao greatly reduced infection by two oomycete pathogens, Phytophthora tropicalis and Phytophthora palmivora, which cause black pod disease. Lesion size and pathogen growth were reduced by up to 85%. Resistance was not conferred by proteins lacking a secretory leader, by proteins with mutations in their PI3P‐binding site, or by a secreted PI4P‐binding protein. Stably transformed, transgenic T. cacao plants expressing two different PI3P‐binding proteins showed substantially enhanced resistance to both P. tropicalis and P. palmivora, as well as to the fungal pathogen Colletotrichum theobromicola. These results demonstrate that secretion of PI3P‐binding proteins is an effective way to increase disease resistance in T. cacao, and potentially in other plants, against a broad spectrum of pathogens.     

Development and Validation of Conventional and Quantitative Polymerase Chain Reaction Assays for the Detection of Storage Rot Potato Pathogens, Phytophthora erythroseptica, Pythium ultimum and Phoma foveata

The diseases pink rot, watery wound rot and gangrene are important storage rot diseases of potato associated predominantly with Phytophthora erythroseptica (P.), Pythium ultimum (Py.) and Phoma exigua (Phoma) var. foveata respectively. Reliable molecular‐based diagnostic tests are required that will not only allow unequivocal identification of symptoms but will further advance epidemiological studies of these potato diseases to increase our understanding and contribute to more effective management and control strategies to the potato industry. Primers and probes were designed in specific regions of the internal transcribed spacer (ITS) regions to develop conventional and real‐time quantitative polymerase chain reaction (PCR) assays able to detect all possible fungal and oomycete pathogens causing pink rot, watery wound rot and gangrene. The specificity of each diagnostic assay was rigorously tested with over 500 fungal/oomycete plant pathogen isolates from potato and reference culture collections, and both conventional and real‐time PCR methods produced similar results. In terms of sensitivity, the detection limits for real‐time PCR went below ag DNA levels compared with pg DNA levels with conventional PCR. The real‐time PCR assays developed to detect Phoma foveata and Py. ultimum on tubers were suitable for the comparative C_t method (ΔΔCt) of quantification using the cytochrome oxidase gene of potato as a normalizer assay; an advantage as the need for a standard curve is eliminated. Each assay detected Phoma species (var. foveata or exigua) from naturally infected tubers showing symptoms of gangrene, and P. erythroseptica or Py. ultimum were also detected following inoculation of Russet Burbank tubers. Each diagnostic assay developed could reliably detect and distinguish between the pink rot, watery wound rot and gangrene‐causing potato pathogens.     

Biotic interactions between aquatic,aero-aquatic and terrestrial hyphomycetes in microcosm

The duplex coefficient of specific correlation was applied for the first time for the estimation of possible interaction between water and terrestrial hyphomycetes (Alternaria sp. and Fusarium sp.) and oomycete Achlya sp. under microcosm conditions. Values of the either direct or indirect coefficient for correlation between water and terrestrial hyphomycetes and oomycete Achlya sp. were predominantly negative. Values of the coefficient for correlation between more frequent water hyphomycete species were more often positive or near 0. Rare water hyphomycete species had usually more or less distinct negative correlation with other water hyphomycete species.     

Subcellular targeting of an evolutionarily conserved plant defensin MtDef4.2 determines the outcome of plant–pathogen interaction in transgenic Arabidopsis

The Medicago truncatula gene encoding an evolutionarily conserved antifungal defensin MtDef4.2 was cloned and characterized. In silico expression analysis indicated that MtDef4.2 is expressed in many tissues during the normal growth and development of M. truncatula. MtDef4.2 exhibits potent broad‐spectrum antifungal activity against various Fusarium spp. Transgenic Arabidopsis thaliana lines in which MtDef4.2 was targeted to three different subcellular compartments were generated. These lines were tested for resistance to the obligate biotrophic oomycete Hyaloperonospora arabidopsidis Noco2 and the hemibiotrophic fungal pathogen Fusarium graminearum PH‐1. MtDef4.2 directed to the extracellular space, but not to the vacuole or retained in the endoplasmic reticulum, conferred robust resistance to H. arabidopsidis. Siliques of transgenic Arabidopsis lines expressing either extracellularly or intracellularly targeted MtDef4.2 displayed low levels of resistance to F. graminearum, but accumulated substantially reduced levels of the mycotoxin deoxynivalenol. The data presented here suggest that extracellularly targeted MtDef4.2 is sufficient to provide strong resistance to the biotrophic oomycete, consistent with the extracellular lifestyle of this pathogen. However, the co‐expression of extracellular and intracellular MtDef4.2 is probably required to achieve strong resistance to the hemibiotrophic pathogen F. graminearum which grows extracellularly and intracellularly.     

Activity profiling of vacuolar processing enzymes reveals a role for VPE during oomycete infection

Vacuolar processing enzymes (VPEs) are important cysteine proteases that are implicated in the maturation of seed storage proteins, and programmed cell death during plant–microbe interactions and development. Here, we introduce a specific, cell‐permeable, activity‐based probe for VPEs. This probe is highly specific for all four Arabidopsis VPEs, and labeling is activity‐dependent, as illustrated by sensitivity for inhibitors, pH and reducing agents. We show that the probe can be used for in vivo imaging and displays multiple active isoforms of VPEs in various tissues and in both monocot and dicot plant species. Thus, VPE activity profiling is a robust, simple and powerful tool for plant research for a wide range of applications. Using VPE activity profiling, we discovered that VPE activity is increased during infection with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa). The enhanced VPE activity is host‐derived and EDS1‐independent. Sporulation of Hpa is reduced on vpe mutant plants, demonstrating a role for VPE during compatible interactions that is presumably independent of programmed cell death. Our data indicate that, as an obligate biotroph, Hpa takes advantage of increased VPE activity in the host, e.g. to mediate protein turnover and nutrient release.     

Diacylglycerol kinases activate tobacco NADPH oxidase‐dependent oxidative burst in response to cryptogein

Cryptogein is a 10 kDa protein secreted by the oomycete Phytophthora cryptogea that activates defence mechanisms in tobacco plants. Among early signalling events triggered by this microbial‐associated molecular pattern is a transient apoplastic oxidative burst which is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity of the RESPIRATORY BURST OXIDASE HOMOLOG isoform D (RBOHD). Using radioactive [³³P]‐orthophosphate labelling of tobacco Bright Yellow‐2 suspension cells, we here provide in vivo evidence for a rapid accumulation of phosphatidic acid (PA) in response to cryptogein because of the coordinated onset of phosphoinositide‐dependent phospholipase C and diacylglycerol kinase (DGK) activities. Both enzyme specific inhibitors and silencing of the phylogenetic cluster III of the tobacco DGK family were found to reduce PA production upon elicitation and to strongly decrease the RBOHD‐mediated oxidative burst. Therefore, it appears that PA originating from DGK controls NADPH‐oxidase activity. Amongst cluster III DGKs, the expression of DGK5‐like was up‐regulated in response to cryptogein. Besides DGK5‐like is likely to be the main cluster III DGK isoform silenced in one of our mutant lines, making it a strong candidate for the observed response to cryptogein. The relevance of these results is discussed with regard to early signalling lipid‐mediated events in plant immunity.