Specific interaction of an RNA-binding protein with the 3′-UTR of its target mRNA is critical to oomycete sexual reproduction

Sexual reproduction is an essential stage of the oomycete life cycle. However, the functions of critical regulators in this biological process remain unclear due to a lack of genome editing technologies and functional genomic studies in oomycetes. The notorious oomycete pathogen Pythium ultimum is responsible for a variety of diseases in a broad range of plant species. In this study, we revealed the mechanism through which PuM90, a stage-specific Puf family RNA-binding protein, regulates oospore formation in P. ultimum. We developed the first CRISPR/Cas9 system-mediated gene knockout and in situ complementation methods for Pythium. PuM90-knockout mutants were significantly defective in oospore formation, with empty oogonia or oospores larger in size with thinner oospore walls compared with the wild type. A tripartite recognition motif (TRM) in the Puf domain of PuM90 could specifically bind to a UGUACAUA motif in the mRNA 3′ untranslated region (UTR) of PuFLP, which encodes a flavodoxin-like protein, and thereby repress PuFLP mRNA level to facilitate oospore formation. Phenotypes similar to PuM90-knockout mutants were observed with overexpression of PuFLP, mutation of key amino acids in the TRM of PuM90, or mutation of the 3′-UTR binding site in PuFLP. The results demonstrated that a specific interaction of the RNA-binding protein PuM90 with the 3′-UTR of PuFLP mRNA at the post-transcriptional regulation level is critical for the sexual reproduction of P. ultimum.

Effect of light and darkness on the growth and development of downy mildew pathogen Hyaloperonospora arabidopsidis

SummaryDisease development in plants requires a susceptible host, a virulent pathogen, and a favourable environment. Oomycete pathogens cause many important diseases and have evolved sophisticated molecular mechanisms to manipulate their hosts. Day length has been shown to impact plant-oomycete interactions but a need exists for a tractable reference system to understand the mechanistic interplay between light regulation, oomycete pathogen virulence, and plant host immunity. Here we present data demonstrating that light is a critical factor in the interaction between Arabidopsis thaliana and its naturally occurring downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). We investigated the role of light on spore germination, mycelium development, sporulation and oospore formation of Hpa, along with defence responses in the host. We observed abundant Hpa sporulation on compatible Arabidopsis under day lengths ranging from 10 to 14 hours. Contrastingly, exposure to constant light or constant dark suppressed sporulation. Exposure to constant dark suppressed spore germination, mycelial development and oospore formation. Interestingly, exposure to constant light stimulated spore germination, mycelial development and oospore formation. A biomarker of plant immune system activation was induced under both constant light and constant dark. Altogether, these findings demonstrate that Hpa has the molecular mechanisms to perceive and respond to light and that both the host and pathogen responses are influenced by the light regime. Therefore, this pathosystem can be used for investigations to understand the molecular mechanisms through which oomycete pathogens like Hpa perceive and integrate light signals, and how light influences pathogen virulence and host immunity during their interactions.

Mating types of Phytophthora colocasiae on the island of Upolu, Samoa

Taro leaf blight (TLB) caused by Phytophthora colocasiae is a damaging disease that destroyed Samoa’s taro industry following its introduction in 1993. The aim of this study was to determine the occurrence of the A1 and A2 mating types of P. colocasiae for a more comprehensive understanding of the risk the pathogen poses for the future of the taro industry in Samoa. In September 2015, 54 isolates of P. colocasiae were collected from taro leaf blight lesions from 13 farms around the island of Upolu, Samoa. The mating types of each isolate was determined by observation of oospore formation when paired with tester isolates of Phytophthora nicotianae of known mating types (A1 or A2). Fifty isolates were found to be A2 mating type and four did not form oospores with either mating type. No A1 or self-fertile isolates were found. These results suggest that the A1 mating type has not been introduced to the island of Upolu, preventing the formation of oospores between compatible mating types of P. colocasiae and lessening the risk of new and potentially more threatening genotypes of the pathogen from emerging through genetic recombination. Keywords taro leaf blight, Colocasia esculenta, taro, sexual reproduction

Baseline Sensitivities of New Fungicides and Their Toxicity to Selected Life Stages of Phytophthora Species from Citrus in California

Phytophthora citrophthora, P. syringae, P. nicotianae, and P. hibernalis are important pathogens of citrus in California but few chemical treatments are currently available. In vitro toxicities of four new fungicides to isolates of Phytophthora spp. from California were determined. Mean effective concentration values to inhibit mycelial growth by 50% for ethaboxam, fluopicolide, mandipropamid, oxathiapiprolin, and mefenoxam were 0.068, 0.04, 0.004, 0.0003, and 0.039 µg/ml, respectively, for 62 isolates of P. citrophthora; 0.005, 0.045, 0.003, 0.0001, and 0.008 µg/ml, respectively, for 71 isolates of P. syringae; 0.016, 0.057, 0.005, 0.0005, and 0.183 µg/ml, respectively, for 31 isolates of P. nicotianae; and 0.030, 0.018, 0.005, <0.0003, and ≤0.001 µg/ml, respectively, for two isolates of P. hibernalis. Mean values for ≥90% inhibition of sporangia formation of four isolates of P. citrophthora were 0.1, 0.28, 0.026, 0.005, and 55 µg/ml for ethaboxam, fluopicolide, mandipropamid, oxathiapiprolin, and mefenoxam, respectively. Zoospore cyst germination of P. citrophthora was most inhibited by oxathiapiprolin and mandipropamid. Chlamydospore formation of P. nicotianae was most sensitive to oxathiapiprolin, with a mean ≥90% reduction (EC>90) of 0.002 µg/ml, moderately sensitive to mandipropamid (EC>90 = 0.2 µg/ml) and mefenoxam (EC>90 = 0.6 µg/ml), and least sensitive to ethaboxam and fluopicolide (EC>90 = 1 µg/ml). Oospore formation of P. nicotianae was inhibited by ≥90% using oxathiapiprolin at 0.0004 µg/ml, mandipropamid at 0.02 µg/ml, ethaboxam at 0.1 µg/ml, or fluopicolide at 0.4 µg/ml, whereas 62% inhibition was obtained by mefenoxam at 40 µg/ml.

Differences in Virulence and Sporulation of Phytophthora kernoviae Isolates Originating From Two Distinct Geographical Regions

Phytophthora kernoviae has only been isolated from the United Kingdom and New Zealand. To understand what differences may exist between isolates from these two distinct geographical regions, virulence studies on three host plants and sporulation on host leaves were conducted on select isolates. Three host plant species (Rhododendron ponticum, Magnolia stellata, and Annona cherimola) were inoculated individually with sporangia of six different isolates from each geographical region. Results showed an overall higher virulence on all three hosts from isolates originating from the United Kingdom. After inoculation, P. kernoviae sporangia and oospore formation on different host leaves were observed and compared with P. cactorum and P. syringae. Results were host dependent, with P. kernoviae producing generally similar or higher amounts of both propagules compared with the other U.S. indigenous species. These results have implications for regulatory agencies and scientists who are interested in preventing its entrance into the United States and learning more about its potential spread.

An RNA symbiont enhances heat tolerance and secondary homothallism in the oomycete Phytophthora infestans

Some strains of Phytophthora infestans, the potato late blight pathogen, harbour a small extrachromosomal RNA called PiERE1. A previous study reported that this RNA symbiont does not noticeably affect its host. Here it is revealed that PiERE1 exerts subtle effects on P. infestans, which result in greater thermotolerance during growth and an increase in secondary homothallism, i.e. oospore formation in the absence of the opposite mating type. The interaction can be considered mutualistic since these traits may increase the fitness of P. infestans in nature. Assays of biomarkers for cellular stress revealed that an Hsp70 chaperone was upregulated by PiERE1. A genome-wide search for more members of the Hsp70 family identified ten belonging to the DnaK subfamily, one in the Hsp110/SSE subfamily, and pseudogenes. Four DnaK subfamily genes encoding predicted cytoplasmic or endoplasmic reticulum proteins were upregulated in strains harbouring PiERE1. This may explain the greater thermotolerance conferred by the RNA element, and suggests that Hsp70 may be a useful biomarker for testing organisms for the cellular effects of symbiotic elements.

First Report of Solanum physalifolium as a Host Plant for Phytophthora infestans in Sweden

In the early summer of 2003, lesions resembling those caused by Phytophthora infestans (Mont.) de Bary on potato were observed on Solanum physalifolium Rusby var. nitidibaccatum (Bitter) Edmonds (2) that was growing as a weed in a parsnip (Pastinaca sativa) field in southern Sweden. When infected leaves of S. physalifolium were observed under the microscope (×200 magnification), sporangia with the same shape and size as those of P. infestans were observed. Pieces of infected leaves of S. physalifolium were put under tuber slices of S. tuberosum (cv. Bintje) in petri dishes and kept at 20°C. After 4 days, mycelium grew through the slices and sporulated profusely. The sporangia on the slices were of the same shape and size as those observed on the infected S. physalifolium leaves. In Sweden, the ratio of A1 and A2 mating types of P. infestans is 50:50, and oospores are commonly found in infected potato crops (1), so isolates from S. physalifolium were tested for mating type by growing them together with reference isolates of a known mating type on agar plates. Nine of the tested isolates were A1 mating type and six were A2 mating type. One self-fertile isolate was found. Naturally infected leaves of S. physalifolium were incubated at 20°C at 100% relative humidity so the lesions could coalesce and to facilitate oospore formation. After 5 days, oospores identical to those of P. infestans were observed under the microscope (×200 magnification). Sporangia produced by isolates originating from S. physalifolium and S. tuberosum were harvested, and a suspension containing 104 sporangia per ml from each isolate was prepared. Five leaves each of S. nigrum, S. physalifolium, and S. tuberosum (cv. Bintje), were inoculated with 10 μl of each sporangial suspension. Inoculated leaves were incubated in sealed petri dishes at 15°C. After 4 days, all S. tuberosum leaves were infected. After 7 days, two of five leaves of S. physalifolium inoculated with the S. tuberosum isolate and two of five S. physalifolium leaves inoculated with the isolate from S. physalifolium were infected. All lesions produced sporangia similar to those formed by P. infestans. S. nigrum was not infected by any of the isolates. The ability of S. physalifolium to act as a host plant for P. infestans producing sporangia during the growing season and oospores for survival between growing seasons may increase the problems of controlling late blight in potato in Sweden. References: (1) J. Dahlberg et al. Field survey of oospore formation by Phytophthora infestans. (Poster Abstr.) Pages 134–135 In: Late Blight: Managing the Global Threat. Proc Global Late Blight Conf. Charlotte Lizarraga, ed. Centro Internacional De La Papa, On-line publication, ISBN 929060-215-5, 2002. (2) J. M. Edmonds. Bot. J. Linn. Soc. 92:1, 1986.