Stage‐specific gene expression during sexual development in Phytophthora infestans

2003

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A gene from Phytophthora infestans that was previously identified as being induced during the development of sexual spores was also found to be active during asexual sporulation. The gene, M90, was expressed as a 3.1-kb primary transcript containing two introns and was predicted to encode a member of the Puf family of translational regulators. The protein showed up to 51% amino acid identity to other Puf proteins within its 353-amino-acid RNA-binding domain. Little similarity extended beyond this region, as noted for other members of the family. Expression of M90 was measured by using RNA blots and transformants of P. infestans expressing a fusion between the M90 promoter and the ␤-glucuronidase (GUS) gene. A 1.3-kb promoter fragment conferred the normal M90 pattern of expression to the GUS reporter in transformants. In matings, expression was first detected in male and female gametangial initials and persisted in mature oospores. Expression was also observed in hyphal tips just prior to asexual sporulation, in sporangiophores, in mature sporangia, and in zoospores. The signal quickly disappeared once spores made the transition to hyphae after germination. Nutrient limitation did not induce the gene. Potential roles for a translational regulator during both sexual development and asexual sporulation are discussed.Mating, sexual sporulation, and asexual sporulation are central to the life histories of many lower eukaryotes. Mating is important because this increases genetic fitness and diversity and because sexual spores are often thick walled and thus more durable than vegetative structures. Also, both sexual and asexual spores are the main agents of dispersal for many species. Detailed molecular analyses of these pathways have been executed only for selected groups, particularly the cellular slime molds, true fungi such as ascomycetes and basidiomycetes, and green algae (2,5,10,13,31). Very limited information is available for most other lower eukaryotes, including oomycetes.Most oomycetes, which include important parasites of plants and animals, undergo both sexual and asexual sporulation. Despite their generally filamentous, fungus-like pattern of growth, oomycetes are diploid and share a close taxonomic relationship with chrysophytes, diatoms, and brown algae rather than with true fungi (3). One of the best-known oomycetes is Phytophthora infestans, which caused the Irish potato famine in the 1840s. The late blight diseases caused by P. infestans continue to have devastating effects on agricultural production and threaten food security (12).The physiology of sporulation in oomycetes is well described, but little is known at a molecular level. In heterothallic Phytophthora, sexual development occurs in response to hormonal interactions between A1 and A2 mating types (4,16,19). Male and female gametangia termed antheridia and oogonia form, and then oogonia penetrate antheridia and develop into oospores. Meiosis occurs in both gametangia, after which haploid antheridial and oogonial nuclei fuse to form a zygote...

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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A Gene Expressed during Sexual and Asexual Sporulation in Phytophthora infestans Is a Member of the Puf Family of Translational Regulators

Cvitanich

2003

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“…The strains shown are a transformant expressing GUS from C3-1400::GUS, two strains with C3-254::GUS, and two strains with C3-151::GUS. (17). Native M82 exhibits low expression in hyphae, strong expression in mating cultures, and very low expression in asexual sporangia and cleaving sporangia.…”

Section: Fig 2 Deletion Analyses Of Pinifc Promoters (A) Arrangemementioning

confidence: 99%

Activation of Zoosporogenesis-Specific Genes in Phytophthora infestans Involves a 7-Nucleotide Promoter Motif and Cold-Induced Membrane Rigidity

Tani

2006

Infections of plants by the oomycete Phytophthora infestans typically result from zoospores, which develop from sporangia at cold temperatures. To help understand the relevant cold-induced signaling pathway, factors regulating the transcription of the zoosporogenesis-specific NIF (nuclear LIM-interactor-interacting factor) gene family were examined. Sequences required for inducing PinifC3 were identified by analyzing truncated and mutated promoters using the ␤-glucuronidase reporter in stable transformants. A 7-nucleotide (nt) sequence located 139 bases upstream of the major transcription start point (GGACGAG) proved essential for the induction of PinifC3 when sporangia were shifted from ambient to cold temperatures. The motif, named the cold box, also conferred cold inducibility to a promoter normally activated only during sexual development. An identical motif was detected in the two other zoosporogenesis-specific NIF genes from P. infestans and three Phytophthora sojae orthologues, and a closely related sequence was found in Phytophthora ramorum orthologues. The 7-nt motif was also found in the promoters of other zoosporogenesis-induced genes. The presence of a cold box-interacting protein in nuclear extracts of P. infestans sporangia was demonstrated using electrophoretic mobility shift assays. Furthermore, zoospore release and cold box-regulated transcription were stimulated by the membrane rigidizer dimethyl sulfoxide and inhibited by the membrane fluidizer benzyl alcohol. The data therefore delineate a pathway in which sporangia perceive cold temperatures through membrane rigidity, which activates signals that drive both zoosporogenesis and cold-box-mediated transcription.

“…Also spotted were cDNAs from actin (actA) and elongation factor 1␣ (EF1) plus controls for nonspecific hybridization. Membranes were treated with 0.4 M NaOH for 10 min and with 3ϫ SSPE (540 mM NaCl, 30 mM NaHPO 4 , 3 mM EDTA, pH 7.4) for 10 min, UV cross-linked to 1,200 J/cm 2 , boiled for 1 min in 0.1% sodium dodecyl sulfate, and then hybridized with 32 P-labeled cDNA as described previously (15). Primary screens for genes upregulated in sporangia were performed using replicate filters, with each clone represented once.…”

Section: Vol 2 2003 Sporangium-specific Gene Expression In P Infesmentioning

confidence: 99%

Sporangium-Specific Gene Expression in theOomycete Phytopathogen Phytophthorainfestans

Kim

2003

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The oomycete genus Phytophthora includes many of the world's most destructive plant pathogens, which are generally disseminated by asexual sporangia. To identify factors relevant to the biology of these propagules, genes induced in sporangia of the potato late blight pathogen Phytophthora infestans were isolated using cDNA macroarrays. Of ϳ1,900 genes known to be expressed in sporangia, 61 were up-regulated >5-fold in sporangia versus hyphae based on the arrays, including 17 that were induced >100-fold. A subset were also activated by starvation and in a nonsporulating mutant. mRNAs of some genes declined in abundance after germination, while others persisted through the germinated zoospore cyst stage. Functions were predicted for about three-quarters of the genes, including potential regulators (protein kinases and phosphatases, transcription factors, and G-protein subunits), transporters, and metabolic enzymes. Predominant among the last were several dehydrogenases, especially a highly expressed sorbitol dehydrogenase that accounted for 3% of the mRNA. Sorbitol dehydrogenase activity also rose during sporulation and several stress treatments, paralleling the expression of the gene. Another interesting metabolic enzyme resembled creatine kinases, which previously were reported only in animals and trypanosomes. These results provide insight into the transcriptional and cellular processes occurring in sporangia and identify potential targets for crop protection strategies.Sporulation is central to the life cycles of most plant-pathogenic lower eukaryotes, including true fungi and oomycetes. The latter group encompasses important pathogens, such as Phytophthora, Pythium, and the downy mildews, which infect many economically significant hosts (14). The molecular biology of sporulation has been characterized in detail for several true fungi (1,11,28,43). However, little information exists on oomycetes, which despite their "fungus-like" appearance are more closely related to organisms such as diatoms and brown algae (3).Asexual sporangia play the major role in spreading Phytophthora infestans, which causes late blight of potato and tomato (17). The roles of nutrients, pH, aeration, light, and humidity in sporulation are well described (33,41). In older portions of plant lesions and in laboratory cultures, sporangia form upon aerial sporangiophores branched from hyphae. In some members of the genus, such as P. infestans, this occurs spontaneously as cultures age, but in others significant sporulation requires washing nutrients from media (41). In both cases, nuclear divisions rapidly occur within sporangiophores, and then nuclei and cytoplasm quickly flow into terminal swellings, which develop into sporangia (7, 32). These mature by forming a basal plug, which delimits the multinucleate sporangium from the coenocytic hypha, and an apical papilla, which is a future site of germination. Two modes of germination are possible in most oomycetes, including P. infestans. Indirect germination by way of zoospores predominates ...