Abstract: Elicitins, extracellular proteins from Phytophthora fungi, elicit a hypersensitivity response (HR), including systemic acquired resistance, in some plants. The elicitin capsicein (~10 kDa) was purified by FPLC from culture filtrates of P. capsici. Purified native and recombinant capsicein induced a hypersensitive response in leaves of the non-host plants Nicotiana glutinosa and Brassica rapa subsp. pekinensis. To search for candidate capsicein-interacting proteins from N. glutinosa, a yeast two-hybrid assay was used. We identified a protein interactor that is homologous to a serine/threonine kinase of the plant receptor-like kinase (RLK) group and designated it NgRLK1. The ORF of NgRLK1 encodes a polypeptide of 832 amino acids (93,490 Da). A conserved domain analysis revealed that NgRLK1 has structural features typical of a plant RLK. NgRLK1 was autophosphorylated, with higher activity in the presence of Mn2+ than Mg2+.
Abstract: Six unique expressed sequence tag (EST) libraries were generated from four developmental stages of Phytophthora sojae P6497. RNA was extracted from mycelia, swimming zoospores, germinating cysts, and soybean (Glycine max
(L.) Merr.) cv. Harosoy tissues heavily infected with P. sojae. Three libraries were created from mycelia growing on defined medium, complex medium, and nutrient-limited medium. The 26,943 high-quality sequences obtained clus-
tered into 7,863 unigenes composed of 2,845 contigs and 5,018 singletons. The total number of P. sojae unigenes matching sequences in the genome assembly was 7,412 (94%). Of these unigenes, 7,088 (90%) matched gene models predicted from the P. sojae sequence assembly, but only 2,047 (26%) matched P. ramorum gene models. Analysis of EST frequency from different growth conditions and morphological stages revealed genes that were specific to or highly represented in particular growth conditions and life stages. Additionally, our results indicate that, during infection, the pathogen derives most of its carbon and energy via glycolysis of sugars in the plant. Sequences identified with putative roles in pathogenesis included avirulence homologs possessing the RxLR motif, elicitins, and hydrolytic enzymes. This large collection of P. sojae ESTs will serve as a valuable public genomic resource.
Abstract: Elicitins, small proteins secreted by Phytophthora and Pythium spp., display the ability to induce plant resistance toward pathogens. Ultra-
structural investigations of cryptogein-treated tobacco plants evidenced host defense responses such as (i) formation of a calcium pectate gel in
intercellular spaces of parenchymas, (ii) impregnation of pectin by phenolic compounds in intercellular spaces of phloem bundles, and (iii) accumulation of phloem proteins (P proteins) in the lumen of leaf sieve elements. These cytological modifications lead to the enhancement of physical barriers that prevent pathogen ingress and restrict host tissue colonization when cryptogein-treated tobacco plants were challenged with the pathogen Phytophthora parasitica. Wall appositions also were observed at most sites of penetration of hyphae. Moreover, growing hyphae exhibited severe morphological damages, suggesting a modified toxic environment. The same induction of P proteins in mature sieve tubes of tobacco leaves was obtained with oligandrin treatment, another elicitin. Cryptogein or oligandrin treatment prevented symptom expression in phytoplasma-infected tobacco plants in contrast with nontreated tobacco plants. Moreover, P protein plugs and occlusion of pore sites by callose were evidenced in sieve elements of treated plants. Both these phloem modifications might prevent the in planta movement of phloemrestricted microorganisms.
Abstract: Elicitins form a family of structurally related proteins that induce the hypersensitive response (HR) in plants, particularly Nicotiana spp. The elicitin family is composed of several classes. Most species of the plant-pathogenic oomycete genus Phytophthora produce the well-characterized 10-kDa canonical elicitins (class I), such as INF1 of the potato and tomato pathogen Phytophthora infestans. Two genes, inf2A and inf2B, encoding a distinct class (class III) of elicitinlike proteins, also occur in P.infestans. Unlike secreted class I elicitins, class III elicitins are thought to be cell-surface-anchored polypeptides. Molecular characterization of the inf2 genes indicated that they are widespread in Phytophthora spp. and occur as a small gene family. In addition, Southern blot and Northern blot hybridizations using gene-specific probes showed that inf2A and inf2B genes and transcripts can be detected in 17 different P. infestans isolates. Functional secreted expression in plant cells of the elicitin domain of the inf1 and inf2 genes was conducted using a binary Potato virus X (PVX) vector (agroinfection) and Agrobacterium tumefaciens transient transformation assays (agroinfiltration), and resulted in HR-like necrotic symptoms and induction of defense response genes in tobacco. However, comparative analyses of elicitor activity of INF1, INF2A, and INF2B revealed significant differences in intensity, specificity, and consistency of HR induction. Whereas INF1 induced the HR in Nicotiana benthamiana, INF2A induced weak symptoms and INF2B induced no symptoms on this plant. Nonetheless, similar to INF1, HR induction by INF2A in N. benthamiana required the ubiquitin ligase-associated protein SGT1. Overall, these results suggest that variation in the resistance of Nicotiana spp. to P. infestans is shadowed by variation in the response to INF elicitins. The ability of tobacco, but not N. benthamiana, to respond to INF2B could explain differences in resistance to P. infestans observed for these two species.
Abstract: Late blight caused by Phytophthora infestans is one of the most
devastating diseases in potato cultivation and is mostly controlled by the application of chemicals. However, introduction of combinations of resistance (R) genes conferring broad-spectrum resistance from wild Solanum species into cultivated potatoes is considered the most practical and promising approach to achieve durable resistance. This can be realized via classical breeding or genetic modification (GM). Because classical breeding is very time-consuming and is often hampered by linkage drag, a GM approach seems logic in this heterozygous and vegetatively propagated crop. During the last decades, many R genes have been identified in several wild Solanum species. Some have been cloned and more will follow. When these genes are derived from species crossable with cultivated potato (socalled cisgenes), application in resistance breeding using a GM approach is similar to an introgression breeding approach, in that the exploited genes are indigenous to the crop. Pending deregulation or derogation of cisgenesis, the use of cisgenic R genes would be an ideal strategy to accomplish durable resistance in potato.
Abstract: Parasitic and pathogenic lifestyles have evolved repeatedly in eukaryotes (93). Several parasitic eukaryotes represent deep phylogenetic lineages, suggesting that they feature unique molecular processes for infecting their hosts. One such group is formed by the oomycetes. Traditionally, due to their filamentous growth habit, oomycetes have been classified in the kingdom Fungi. However, modern molecular and biochemical analyses suggest that oomycetes have little taxonomic affinity with filamentous fungi but are more closely related to brown algae (heterokonts) in the Stramenopiles, one of several major eukaryotic kingdoms (2, 56, 93). The most notable and beststudied oomycete species is Phytophthora infestans, the Irish famine pathogen. P. infestans causes late blight, a devastating and reemerging disease of potatoes and tomatoes (4, 17, 18, 82, 90–92). Other oomycetes include destructive plant and animal pathogens, as well as saprophytes that are beneficial to the environment (56). Despite their peculiar phylogenetic affinities and economic importance, oomycetes were chronically understudied at the molecular level. However, in recent years, increased awareness of the evolutionary history of oomycetes as unique eukaryotic microbes resulted in the emergence of a group of researchers specializing in oomycete genetics. The evolution of this community has been driven by the biological properties of oomycetes, which require alternative methodologies. Technical developments, such as routine DNA transformation, use of reporter genes, and genetic manipulation using gene silencing have facilitated the discovery and functional analyses of several interesting genes. With recent efforts in genomics and functional genomics and the resulting resources, genetic research on oomycetes has entered an exciting phase. This review provides an overview of oomycete biology, with a particular emphasis on molecular genetics. It describes the molecular and genomic resources available for these organisms and discusses the current status of, and future perspectives in, basic studies of pathogenic oomycetes. Unfortunately, too little is known about the molecular genetics of the pathogenicity of oomycetes
other than Phytophthora to warrant a comprehensive review. Therefore, the section on infection mechanisms focuses on Phytophthora. For additional information on various aspects of oomycete biology, the reader is referred to a number of recent reviews (4, 38, 39, 43, 82, 91, 92, 99, 100).
Abstract: Two new crosses involving four races (races 7, 16, 17, and 25) of the soybean root and stem rot pathogen Phytophthora sojae were established (7/16 cross; 17/25 cross). An F2 population derived from each cross was used to determine the genetic basis of avirulence towards 11 different resistance genes in soybean. Avirulence was found to be dominant and determined by a single locus for Avr1b, 1d, 1k, 3b, 4, and 6, as expected for a simple gene-for-gene model. We also observed several cases of segregation, inconsistent with a single dominant gene being solely responsible for avirulence, which suggests that the genetic background of the different crosses can affect avirulence. Avr4 and 6 cosegregated in both the 7/16 and 17/25 crosses and, in the 7/16 cross, Avr1b and 1k were closely linked. Information from segregating RAPD, RFLP, and AFLP markers screened on F2 progeny from the two new crosses and two crosses described previously (a total of 212 F2 individuals, 53 from each cross) were used to construct an integrated genetic linkage map of P. sojae. This revised genetic linkage map consists of 386 markers comprising 35 RFLP, 236 RAPD, and 105 AFLP markers, as well as 10 avirulence genes. The map is composed of 21 major linkage groups and seven minor linkage groups covering a total map distance of 1640.4 cM.
Abstract: The genus Phytophthora belongs to the oomycetes in the eukaryotic stramenopile lineage and is comprised of over 65 species that are all destructive plant pathogens on a wide range of dicotyledons. Phytophthora produces elicitins (ELIs),a group of extracellular elicitor proteins that cause a hypersensitive response in tobacco. Database mining revealed several new classes of elicitin-like (ELL) sequences with diverse elicitin domains in Phytophthora infestans, Phytophthora sojae, Phytophthora brassicae, and Phytophthora ramorum. ELIs and ELLs were shown to be unique to Phytophthora and Pythium species. They are ubiquitous among Phytophthora species and belong to one of the most highly conserved and complex protein families in the Phytophthora genus. Phylogeny construction with elicitin domains derived from 156 ELIs and ELLs showed that most of the diversified family members existed prior to divergence of Phytophthora species from a common ancestor. Analysis to discriminate diversifying and purifying selection showed that all 17 ELI and ELL clades are under purifying selection. Within highly similar ELI groups there was no evidence for positively selected amino acids suggesting that purifying selection contributes to the continued existence of this diverse protein
family. Characteristic cysteine spacing patterns were found for each phylogenetic clade. Except for the canonical clade ELI-1, ELIs and ELLs possess C-terminal domains of variable length, many of which have a high threonine, serine, or proline content suggesting an association with the cell wall. In addition, some ELIs and ELLs have a predicted glycosylphosphatidylinositol site suggesting anchoring of the C-terminal domain to the cell membrane. The eli and ell genes belonging to different clades are clustered in the genomes. Overall, eli and ell genes are expressed at different levels and in different life cycle stages but those sharing the same phylogenetic clade appear to have similar expression patterns.
Abstract: The oomycete Phytophthora sojae is a severe pathogen of soybean Several resistance genes against races of P. sojae exist in soybean but the nature of corresponding avirulence genes is unknown. Clones encoding four different isoforms of a protein elicitor from P. sojae (sojein 1-4) belonging to the class of acidic x-elicitins have been isolated. These 87 amino acid proteins show high homology to elicitins from other Phytophthora species. The different sojein isoforms were expressed in Escherichiacoli as His-tagged fusion proteins. Purified sojein as well as recombinant sojein isoforms induce hypersensitive reaction (HR)-like lesions in tobacco but are not active as race-specific elicitors in soybean. However all sojein isoforms induce defence-related genes like those encoding phenylalanine ammonia lyase, glutathione-S-transferase and chalcone synthase in tobacco and soybean plants and cell cultures. It is concluded that sojeins contribute to the induction of defence responses but that they are not involved in race specific recognition of the P. sojae races by soybean plants.
Abstract: We adapted and optimized the use of the Agrobacterium tumefaciens binary PVX expression system (PVX agroinfection)to screen Solanum plants for response to pathogen elicitors and applied the assay to identify a total of 11 clones of Solanum huancabambense and Solanum microdontum, out of 31 species tested, that respond to the elicitins INF1, INF2A and INF2B of
Phytophthora infestans. Prior to this study, response to INF elicitins was only known in Nicotiana spp. within the Solanaceae. The identified
S. huancabambense and S. microdontum clones also exhibited hypersensitivity-like cell death following infiltration with purified recombinant INF1, INF2A and INF2B, thereby validating the screening protocol. Comparison of INF elicitin activity revealed that Nicotiana plants responded to significantly lower concentrations than Solanum, suggesting variable levels of sensitivity to INF elicitins. We exploited natural variation in response to INF elicitins in the identified Solanum accessions to evaluate the relationship between INF recognition and late blight resistance. Interestingly, several INF-responsive Solanum plants were susceptible to P. infestans. Also, an S. microdontum×Solanum tuberosum(potato) population that segregates for INF response was generated but failed to identify a measurable contribution of INF response to resistance. These results suggest that in Solanum, INF elicitins are recognized as general elicitors and do not have a measurable contribution to disease resistance.
Abstract