Cross-species global proteomics reveals conserved and unique processes in Phytophthora sojae and Phytophthora ramorum

Phytophthora ramorum and Phytophthora sojae are destructive plant pathogens. P. sojae has a narrow host range, whereas P. ramorum has a wide host range. A global proteomics comparison of the vegetative (mycelium) and infective (germinating cyst) life stages of P. sojae and P. ramorum was conducted to identify candidate proteins involved in host range, early infection, and vegetative growth. Sixty-two candidates for early infection, 26 candidates for vegetative growth, and numerous proteins that may be involved in defining host specificity were identified. In addition, common life stage proteomic trends between the organisms were observed. In mycelia, proteins involved in transport and metabolism of amino acids, carbohydrates, and other small molecules were up-regulated. In the germinating cysts, up-regulated proteins associated with lipid transport and metabolism, cytoskeleton, and protein synthesis were observed. It appears that the germinating cyst catabolizes lipid reserves through the beta-oxidation pathway to drive the extensive protein synthesis necessary to produce the germ tube and initiate infection. Once inside the host, the pathogen switches to vegetative growth in which energy is derived from glycolysis and utilized for synthesis of amino acids and other molecules that assist survival in the plant tissue.     

A novel Phytophthora infestans haustorium-specific membrane protein is required for infection of potato

Phytophthora infestans causes late-blight, a devastating and re-emerging disease of potato crops. During the early stages of infection, P. infestans differentiates infection-specific structures such as appressoria for host epidermal cell penetration, followed by infection vesicles, and haustoria to establish a biotrophic phase of interaction. Here we report the cloning, from a suppression subtractive hybridization library, of a P. infestans gene called Pihmp1 encoding a putative glycosylated protein with four closely spaced trans-membrane helices. Pihmp1 expression is upregulated in germinating cysts and in germinating cysts with appressoria, and significantly upregulated throughout infection of potato. Transient gene silencing of Pihmp1 led to loss of pathogenicity and indicated involvement of this gene in the penetration and early infection processes of P. infestans. P. infestans transformants expressing a Pihmp1::monomeric red fluorescent protein (mRFP) fusion demonstrated that Pihmp1 was translated in germinating sporangia, germinating cysts and appressoria, accumulated in the appressorium, and was located at the haustorial membrane during infection. Furthermore, we discovered that haustorial structures are formed over a 3 h period, maturing for up to 12 h, and that their formation is initiated only at sites on the surface of intercellular hyphae where Pihmp1::mRFP is localized. We propose that Pihmp1 is an integral membrane protein that provides physical stability to the plasma membrane of P. infestans infection structures. We have provided the first evidence that the surface of oomycete haustoria possess proteins specific to these biotrophic structures, and that formation of biotrophic structures (infection vesicles and haustoria) is essential to successful host colonization by P. infestans.     

Genes expressed in zoospores of<Emphasis Type="Italic"> Phytophthora nicotianae</Emphasis>

The genus Phytophthora includes many highly destructive plant pathogens. In many Phytophthora species, pathogen dispersal and initiation of plant infection are achieved by motile, biflagellate zoospores that are chemotactically attracted to suitable infection sites. In order to study gene expression in zoospores, we have constructed a cDNA library using mRNA from zoospores of Phytophthora nicotianae. The library was arrayed and screened using probes derived from mycelium or zoospore mRNA. More than 400 clones representing genes preferentially expressed in zoospores were identified and sequenced from the 5 end of the insert. The expressed sequence tags (ESTs) generated were found to represent 240 genes. The ESTs were compared to sequences in GenBank and in the Phytophthora Genome Consortium database, and classified according to putative function based on homology to known proteins. To further characterize the identified genes, a colony array was created on replicate nylon filters and screened with probes derived from four Phytophthora developmental stages including zoospores, germinating cysts, vegetative mycelium and sporulating hyphae, and from inoculated and uninoculated tobacco seedlings. Data from sequence analysis and colony array screening were compiled into a local database, and searched to identify genes that are preferentially expressed in zoospores for future functional analysis.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by C. A. M. J. J. van den Hondel     

Cloning and nucleotide sequencing of three heat shock protein genes (hsp90, hsc70, and hsp19.5) from the diamondback moth, Plutella xylostella (L.) and their expression in relation to developmental stage and temperature

Heat shock protein genes, hsp90, hsc70, and hsp19.5, were cloned and sequenced from the diamondback moth, Plutella xylostella (L.) by RT-PCR and RACE method. The cDNA sequence analysis of hsp90 and hsp19.5 revealed open reading frames (ORFs) of 2,151 and 522 bp in length, which encode proteins with calculated molecular weights of 82.4 and 19.5 kDa, respectively. Analysis of cDNA from hsc70 revealed an ORF of 1,878 bp coding a protein with a calculated molecular weight of 69.3 kDa. Furthermore, the analysis of genomic DNA from hsc70 confirmed the presence of introns while no introns were apparent in hsp90 and hsp19.5. Southern blot analysis suggested the presence of multiple copies of each gene family in the DBM genome. Detectable expression of hsp19.5 was observed at the pupal stage while expression of hsp90 and hsc70 was detected at both pupal and adult stages. At adult stage, females showed a higher expression of hsp90 and hsc70 than males. An increased expression was observed in all three genes after exposure to a high temperature in both sexes. These results suggest that in addition to a heat shock response, these HSP genes might be involved in other functions during the course of development in DBM.     

Isolation and characterization of differentially expressed genes in the mycelium and fruit body of Tuber borchii

The transition from vegetative mycelium to fruit body in truffles requires differentiation processes which lead to edible fruit bodies (ascomata) consisting of different cell and tissue types. The identification of genes differentially expressed during these developmental processes can contribute greatly to a better understanding of truffle morphogenesis. A cDNA library was constructed from vegetative mycelium RNAs of the white truffle Tuber borchii, and 214 cDNAs were sequenced. Up to 58% of the expressed sequence tags corresponded to known genes. The majority of the identified sequences represented housekeeping proteins, i.e., proteins involved in gene or protein expression, cell wall formation, primary and secondary metabolism, and signaling pathways. We screened 171 arrayed cDNAs by using cDNA probes constructed from mRNAs of vegetative mycelium and ascomata to identify fruit body-regulated genes. Comparisons of signals from vegetative mycelium and fruit bodies bearing 15 or 70% mature spores revealed significant differences in the expression levels for up to 33% of the investigated genes. The expression levels for six highly regulated genes were confirmed by RNA blot analyses. The expression of glutamine synthetase, 5-aminolevulinic acid synthetase, isocitrate lyase, thioredoxin, glucan 1,3-beta-glucosidase, and UDP-glucose:sterol glucosyl transferase was highly up-regulated, suggesting that amino acid biosynthesis, the glyoxylate cycle pathway, and cell wall synthesis are strikingly altered during morphogenesis.     

Isolation and Characterization of Differentially Expressed Genes in the Mycelium and Fruit Body of Tuber borchii

The transition from vegetative mycelium to fruit body in truffles requires differentiation processes which lead to edible fruit bodies (ascomata) consisting of different cell and tissue types. The identification of genes differentially expressed during these developmental processes can contribute greatly to a better understanding of truffle morphogenesis. A cDNA library was constructed from vegetative mycelium RNAs of the white truffle Tuber borchii, and 214 cDNAs were sequenced. Up to 58% of the expressed sequence tags corresponded to known genes. The majority of the identified sequences represented housekeeping proteins, i.e., proteins involved in gene or protein expression, cell wall formation, primary and secondary metabolism, and signaling pathways. We screened 171 arrayed cDNAs by using cDNA probes constructed from mRNAs of vegetative mycelium and ascomata to identify fruit body-regulated genes. Comparisons of signals from vegetative mycelium and fruit bodies bearing 15 or 70% mature spores revealed significant differences in the expression levels for up to 33% of the investigated genes. The expression levels for six highly regulated genes were confirmed by RNA blot analyses. The expression of glutamine synthetase, 5-aminolevulinic acid synthetase, isocitrate lyase, thioredoxin, glucan 1,3-β-glucosidase, and UDP-glucose:sterol glucosyl transferase was highly up-regulated, suggesting that amino acid biosynthesis, the glyoxylate cycle pathway, and cell wall synthesis are strikingly altered during morphogenesis.