Identification of Avramr1 from Phytophthora infestans using long read and cDNA pathogen-enrichment sequencing (PenSeq)

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, significantly hampers potato production. Recently, a new Resistance to Phytophthora infestans (Rpi) gene, Rpi-amr1, was cloned from a wild Solanum species, Solanum americanum. Identification of the corresponding recognized effector (Avirulence or Avr) genes from P. infestans is key to elucidating their naturally occurring sequence variation, which in turn informs the potential durability of the cognate late blight resistance. To identify the P. infestans effector recognized by Rpi-amr1, we screened available RXLR effector libraries and used long read and cDNA pathogen-enrichment sequencing (PenSeq) on four P. infestans isolates to explore the untested effectors. Using single-molecule real-time sequencing (SMRT) and cDNA PenSeq, we identified 47 highly expressed effectors from P. infestans, including PITG_07569, which triggers a highly specific cell death response when transiently coexpressed with Rpi-amr1 in Nicotiana benthamiana, suggesting that PITG_07569 is Avramr1. Here we demonstrate that long read and cDNA PenSeq enables the identification of full-length RXLR effector families and their expression profile. This study has revealed key insights into the evolution and polymorphism of a complex RXLR effector family that is associated with the recognition by Rpi-amr1.     

中国苹果病害病原菌物名录

苹果为我国主要栽培水果,苹果产业在我国农业生产中占有重要地位。病原菌物是苹果病害的主要病原物,对我国苹果产量和品质造成严重损害。国际上病原菌物为苹果主要病原类型,其数量占苹果病原物的93.4%。我国植物病理学家和菌物学家对苹果病害的病原学进行了长期研究,描述与记载了大量国外已报道的病原真菌和病原卵菌,也描述了一些国外尚未记载的病原菌。随着菌物分类研究的深入、分类系统及菌物命名规则变化等,许多病害的病原名称发生了较大变化,对名称的使用造成了诸多不便,影响了苹果病害相关知识的交流。本文汇总了我国已经描述的苹果菌物病害的病原种类,其中病原真菌149种,病原卵菌6种,病原菌物占苹果病原物种类的90.6%。依据最新分类系统、菌物命名法规和汉语名称规则,对相关病原菌物的拉丁学名、中文名称以及病害汉语名称等进行了整理和修订。该项工作有利于相关植物病理学研究者、植保工作者、园艺工作者、管理人员及基层推广工作者对苹果病原菌物名称的检索和规范使用,促进学术交流和科学普及等。     

Microsatellite markers reveal two admixed genetic groups and an ongoing displacement within the French population of the invasive plant pathogen Phytophthora infestans

Potato late blight is an example of a re‐emerging disease of plants. Phytophthora infestans was first introduced into Europe during the 19th century, where it caused the Irish potato famine. During the 20th century several additional introduction events have been suspected, especially in the mid‐70s due to the import of large quantities of potato needed after the shortage caused by drought in 1976. Here, we investigate the genetic population structure of Phytophthora infestans, at the first stages of a recent invasion process in France. A total of 220 isolates was collected from 20 commercial fields of the potato susceptible cultivar Bintje, during two consecutive years (2004 and 2005). Clustering analyses based on eight recently developed microsatellite markers reveal that French P. infestans populations are made of two differentiated genetic clusters of isolates (F_ST = 0.19). This result suggests multiple introductions of P. infestans into France, either through the introduction of a composite population of isolates or through the successive introduction of isolates having differentiated genetic backgrounds. Both clusters identified have a strong clonal structure and are similar regarding genetic diversity and mating type composition. The maintenance of differentiation between the two genetic clusters should result from the low or non‐existent contribution of sexual reproduction in French P. infestans populations.     

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.     

Pythium vexans causing patch canker of rubber trees on Hainan Island,China

This is the first report of patch canker disease of rubber trees (clone RRIM600) in China. It is characterized by discrete irregular patches of rotted, discolored bark and wood, accompanied by a decrease in latex flow. A total of seven isolates of Pythium vexans were obtained from the diseased bark of the trunks and roots of rubber trees. Inoculating these isolates into healthy, mature rubber trees resulted in symptoms similar to patch canker and the same fungal species was re-isolated from the diseased tissues. This is also the first record of Py. vexans in Hainan.     

Alteration of pathogenicity-linked life-history traits by resistance of its host Solanum tuberosum impacts sexual reproduction of the plant pathogenic oomycete Phytophthora infestans

Although sexual reproduction implies a cost, it represents an evolutionary advantage for the adaptation and survival of facultative sexual pathogens. Understanding the maintenance of sex in pathogens requires to analyse how host resistance will impact their sexual reproduction through the alteration of their life-history traits. We explored this experimentally using potato (Solanum tuberosum) and one of its pathogens, the heterothallic oomycete Phytophthora infestans. Sexual reproduction was highest on hosts favouring asexual multiplication of the pathogen, suggesting similar nutritional requirements for both sexual and asexual sporulation. Sexual reproduction was also highest on hosts decreasing the latent period, probably because of a trade-off between growth and reproduction. Distinguishing host effects on each pathogenic trait remains however uneasy, as most life-history traits linked to pathogenicity were not independent of each other. We argue that sexual reproduction of P. infestans is an adaptation to survive when the host is susceptible and rapidly destroyed.     

Fungal Pathogens: The Battle for Plant Infection

The attempted infection of a plant by a pathogen, such as a fungus or an Oomycete, may be regarded as a battle whose major weapons are proteins and smaller chemical compounds produced by both organisms. Indeed, plants produce an astonishing plethora of defense compounds that are still being discovered at a rapid pace. This pattern arose from a multi-million year, ping-pong?type co-evolution, in which plant and pathogen successively added new chemical weapons in this perpetual battle. As each defensive innovation was established in the host, new ways to circumvent it evolved in the pathogen. This complex co-evolution process probably explains not only the exquisite specificity observed between many pathogens and their hosts, but also the ineffectiveness or redundancy of some defensive genes which often encode enzymes with overlapping activities. Plants evolved a complex, multi-level series of structural and chemical barriers that are both constitutive or preformed and inducible. These defenses may involve strengthening of the cell wall, hypersensitive response (HR), oxidative burst, phytoalexins and pathogenesis-related (PR) proteins. The pathogen must successfully overcome these obstacles before it succeeds in causing disease. In some cases, it needs to modulate or modify plant cell metabolism to its own benefit and/or to abolish defense reactions. Central to the activation of plant responses is timely perception of the pathogen by the plant. A crucial role is played by elicitors which, depending on their mode of action, are broadly classified into nonspecific elicitors and highly specific elicitors or virulence effector/avirulence factors. A protein battle for penetration is then initiated, marking the pathogen attempted transition from extracellular to invasive growth before parasitism and disease can be established. Three major types of defense responses may be observed in plants: non-host resistance, host resistance, and host pathogenesis. Plant innate immunity may comprise a continuum from non-host resistance involving the detection of general elicitors to host-specific resistance involving detection of specific elicitors by R proteins. It was generally assumed that non-host resistance was based on passive mechanisms and that nonspecific rejection usually arose as a consequence of the non-host pathogen failure to breach the first lines of plant defense. However, recent evidence has blurred the clear-cut distinction among non-host resistance, host-specific resistance and disease. The same obstacles are also serious challenges for host pathogens, reducing their success rate significantly in causing disease. Indeed, even susceptible plants mount a (insufficient) defense response upon recognition of pathogen elicited molecular signals. Recent evidence suggests the occurrence of significant overlaps between the protein components and signalling pathways of these types of resistance, suggesting the existence of both shared and unique features for the three branches of plant innate immunity.     

New Insights in the Life Cycle and Epidemics of Phytophthora porri on Leek

White tip, caused by Phytophthora porri, is a devastating disease in the autumn and winter production of leek (Allium porrum) in Europe. This study investigated the disease cycle of P. porri in laboratory and field conditions. Oospores readily germinated in the presence of non‐sterile soil extract at any temperature between 4 and 22°C, with the formation of sporangia which released zoospores. The zoospores survived at least 7 weeks in water at a temperature range of 0 till 24°C. Microscopic examinations revealed that zoospores encysted and germinated on the leek leaf surface and hyphae entered the leaf directly through stomata or by penetrating via appressoria. Oospores were formed in the leaves within 6 days, while sporangia were not produced. By monitoring disease progress in fields with a different cropping history of leek, it could be deduced that P. porri survives in soil for up to 4 years. Disease progress during three consecutive years was correlated with average daily rainfall in the infection period. Disease incidence on leek was reduced when rain splash was excluded by growing the plants in an open hoop greenhouse. Based on these findings, we propose a disease cycle for P. porri in which oospores germinate in puddles, and zoospores reach the leaves by rain splash and survive in water in the leaf axils, from where they infect the plant by direct penetration or via stomata. When conditions become unfavourable, oospores are produced in the leaves which again reach the soil when leaves decay. Secondary spread of the disease by sporangia does not seem to be important.     

Effect of Vitamin E and Its Analogues Having Various Molecular Structures on the Growth and Lipid Composition of Pythium debaryanum

The effect of exogenously added vitamin E and its synthetic analogues (the hydrophilic form of vitamin E and chromans C₁₃and C₁) at a concentration of 9.86 × 10^–5M on the growth, lipogenic activity, and the fatty acid composition of the eicosapolyenoic acid–synthesizing oomycete Pythium debaryanumwas studied. The effect was found to depend on the molecular structure of particular compounds. For instance, vitamin E and chroman C₁₃stimulated fungal growth, whereas chroman C₁inhibited it. The hydrophilic form of vitamin E enhanced the lipogenic activity of the oomycete. The studied compounds, which possess antioxidant activity, did not exert any noticeable effect on the content of eicosapolyenoic acids and the degree of the unsaturation of fungal lipids.     

Mining oomycete proteomes for metalloproteases leads to identification of candidate virulence factors in Phytophthora infestans

Pathogens deploy a wide range of pathogenicity factors, including a plethora of proteases, to modify host tissue or manipulate host defences. Metalloproteases (MPs) have been implicated in virulence in several animal and plant pathogens. Here we investigated the repertoire of MPs in 46 stramenopile species including 37 oomycetes, 5 diatoms, and 4 brown algae. Screening their complete proteomes using hidden Markov models (HMMs) trained for MP detection resulted in over 4,000 MPs, with most species having between 65 and 100 putative MPs. Classification in clans and families according to the MEROPS database showed a highly diverse MP repertoire in each species. Analyses of domain composition, orthologous groups, distribution, and abundance within the stramenopile lineage revealed a few oomycete-specific MPs and MPs potentially related to lifestyle. In-depth analyses of MPs in the plant pathogen Phytophthora infestans revealed 91 MPs, divided over 21 protein families, including 25 MPs with a predicted signal peptide or signal anchor. Expression profiling showed different patterns of MP gene expression during pre-infection and infection stages. When expressed in leaves of Nicotiana benthamiana, 12 MPs changed the sizes of lesions caused by inoculation with P. infestans; with 9 MPs the lesions were larger, suggesting a positive effect on the virulence of P. infestans, while 3 MPs had a negative effect, resulting in smaller lesions. To the best of our knowledge, this is the first systematic inventory of MPs in oomycetes and the first study pinpointing MPs as potential pathogenicity factors in Phytophthora.