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.

Avrova AO ，Boevink PC ，Young V ，Grenville-Briggs LJ ，van West P ，Birch PR ，Whisson SC ... -  《-》

Elevated amino acid biosynthesis in Phytophthora infestans during appressorium formation and potato infection.

Appressorium formation is believed to be an important event in establishing a successful interaction between the late blight pathogen, Phytophthora infestans, and its host plants potato and tomato. An understanding of molecular events occurring in appressorium development could suggest new strategies for controlling late blight. We used parallel studies of the transcriptome and proteome to identify genes and proteins that are up-regulated in germinating cysts developing appressoria. As a result, five distinct genes involved in amino acid biosynthesis were identified that show increased expression in germinating cysts with appressoria. These are a methionine synthase (Pi-met1), a ketol-acid reductoisomerase (Pi-kari1), a tryptophan synthase (Pi-trp1), an acetolactate synthase (Pi-als1), and a threonine synthase (Pi-ts1). Four of these P. infestans genes were also up-regulated, although to lower levels, during the early, biotrophic phase of the interaction in potato and all five were considerably up-regulated during the transition (48 hpi) to the necrotrophic phase of the interaction. Real-time RT-PCR revealed that expression of potato homologues of the amino acid biosynthesis genes increased during biotrophic and necrotrophic infection phases. Furthermore, we investigated levels of free amino acids in the pre-infection stages and found that in most cases there was a decrease in free amino acids in zoospores and germinating cysts, relative to sporangia, followed by a sharp increase in germinating cysts with appressoria. Amino acid biosynthesis would appear to be important for pathogenicity in P. infestans, providing a potential metabolic target for chemical control.

Grenville-Briggs LJ ，Avrova AO ，Bruce CR ，Williams A ，Whisson SC ，Birch PR ，van West P ... -  《FUNGAL GENETICS AND BIOLOGY》

Invertases in Phytophthora infestans Localize to Haustoria and Are Programmed for Infection-Specific Expression.

The oomycete , the causal agent of potato and tomato blight, expresses two extracellular invertases. Unlike typical fungal invertases, the genes are not sucrose induced or glucose repressed but instead appear to be under developmental control. Transcript levels of both genes were very low in mycelia harvested from artificial medium but high in preinfection stages (sporangia, zoospores, and germinated cysts), high during biotrophic growth in leaves and tubers, and low during necrotrophy. Genome-wide analyses of metabolic enzymes and effectors indicated that this expression profile was fairly unusual, matched only by a few other enzymes, such as carbonic anhydrases and a few RXLR effectors. Genes for other metabolic enzymes were typically downregulated in the preinfection stages. Overall metabolic gene expression during the necrotrophic stage of infection clustered with artificial medium, while the biotrophic phase formed a separate cluster. Confocal microscopy of transformants expressing green fluorescent protein (GFP) fusions indicated that invertase protein resided primarily in haustoria during infection. This localization was not attributable to haustorium-specific promoter activity. Instead, the N-terminal regions of proteins containing signal peptides were sufficient to deliver proteins to haustoria. Invertase expression during leaf infection was linked to a decline in apoplastic sucrose, consistent with a role of the enzymes in plant pathogenesis. This was also suggested by the discovery that invertase genes occur across multiple orders of oomycetes but not in most animal pathogens or a mycoparasite. Oomycetes cause hundreds of diseases in economically and environmentally significant plants. How these microbes acquire host nutrients is not well understood. Many oomycetes insert specialized hyphae called haustoria into plant cells, but unlike their fungal counterparts, a role in nutrition has remained unproven. The discovery that invertases localize to haustoria provides the first strong evidence that these structures participate in feeding. Since regions of proteins containing signal peptides targeted proteins to the haustorium-plant interface, haustoria appear to be the primary machinery for secreting proteins during biotrophic pathogenesis. Although oomycete invertases were acquired laterally from fungi, their expression patterns have adapted to the lifestyle by abandoning substrate-level regulation in favor of developmental control, allowing the enzymes to be produced in anticipation of plant colonization. This study highlights how a widely distributed hydrolytic enzyme has evolved new behaviors in oomycetes.

Kagda MS ，Martínez-Soto D ，Ah-Fong AMV ，Judelson HS ... -  《mBio》

Differential recognition of Phytophthora infestans races in potato R4 breeding lines.

Introgression breeding has resulted in several potato lines that are resistant to late blight, a devastating plant disease caused by the oomycete Phytophthora infestans. The traditional differential set consists of potato lines with 11 late blight resistance specificities, referred to as R1 to R11. With the exception of the R4 locus, all the resistance loci in these lines have been genetically mapped or positioned in resistance (R) gene clusters. In this study, we show that potato lines that are defined to carry R4 do not necessarily recognize the same P. infestans strains. Field isolates appeared to be avirulent on either the R4 differential developed by Mastenbroek or the one developed by Black but not on both. Previously, we identified the avirulence gene PiAvr4, which is a member of the RXLR effector family. In planta expression of PiAvr4 revealed that recognition of PiAvr4 is strictly confined to the Mastenbroek R4 differential. Segregation of the trait in two independent F1 progenies showed that late blight resistance in this differential is determined by a single dominant gene, now referred to as R4Ma.

van Poppel PM ，Huigen DJ ，Govers F 《PHYTOPATHOLOGY》

A bZIP transcription factor from Phytophthora interacts with a protein kinase and is required for zoospore motility and plant infection.

Zoospores are critical in the disease cycle of Phytophthora infestans, a member of the oomycete group of fungus-like microbes and the cause of potato late blight. A protein kinase induced during zoosporogenesis, Pipkz1, was shown to interact in the yeast two-hybrid system with a putative bZIP transcription factor. This interaction was confirmed in vitro using a pull-down assay. The transcription factor gene, Pibzp1, was single copy and expressed in all tissues. Transformants of P. infestans stably silenced for Pibzp1 were generated using plasmids expressing its coding region in sense or antisense orientations. A protoplast transformation method induced silencing more efficiently than transformation by an electroporation scheme. Wild-type and silenced strains exhibited no differences in hyphal growth or morphology, mating, sporangia production or zoospore release. However, zoospores from the mutants spun in tight circles, instead of exhibiting the normal pattern of straight swimming punctuated by turns. Zoospore encystment was unaffected by silencing, but cysts germinated more efficiently than controls. Germinated cysts from the mutants failed to develop appressoria and were unable to infect plants; however, they could colonize wounded tissue. These phenotypes indicate that Pibzp1 is a key regulator of several stages of the zoospore-mediated infection pathway.

Blanco FA ，Judelson HS 《MOLECULAR MICROBIOLOGY》