Uptake of oomycete RXLR effectors into host cells by clathrin-mediated endocytosis.

Filamentous (oomycete and fungal) plant pathogens deliver cytoplasmic effector proteins into host cells to facilitate disease. How RXLR effectors from the potato late blight pathogen Phytophthora infestans enter host cells is unknown. One possible route involves clathrin-mediated endocytosis (CME). Transient silencing of NbCHC, encoding clathrin heavy chain, or the endosome marker gene NbAra6 encoding a Rab GTPase in the model host Nicotiana benthamiana, attenuated P. infestans infection and reduced translocation of RXLR effector fusions from transgenic pathogen strains into host cells. By contrast, silencing PP1c isoforms, susceptibility factors not required for endocytosis, reduced infection but did not attenuate RXLR effector uptake. Endosome enrichment by ultracentrifugation and sucrose gradient fractionation revealed co-localization of RXLR effector Pi04314-RFP with clathrin-coated vesicles. Immunopurification of clathrin- and NbAra6-associated vesicles during infection showed that RXLR effectors Pi04314-RFP and AvrBlb1-RFP, but not apoplastic effector PiSCR74-RFP, were co-immunoprecipitated during infection with pathogen strains secreting these effectors. Tandem mass spectrometry analyses of proteins co-immunoprecipitated with NbAra6-GFP during infection revealed enrichment of host proteins associated with endocytic vesicles alongside multiple pathogen RXLR effectors, but not apoplastic effectors, including PiSCR74, which do not enter host cells. Our data show that the uptake of P. infestans RXLR effectors into plant cells occurs via CME.

Draft genome assemblies for tree pathogens Phytophthora pseudosyringae and Phytophthora boehmeriae.

Species of Phytophthora, plant pathogenic eukaryotic microbes, can cause disease on many tree species. Genome sequencing of species from this genus has helped to determine components of their pathogenicity arsenal. Here, we sequenced genomes for two widely distributed species, Phytophthora pseudosyringae and Phytophthora boehmeriae, yielding genome assemblies of 49 and 40 Mb, respectively. We identified more than 270 candidate disease promoting RXLR effector coding genes for each species, and hundreds of genes encoding candidate plant cell wall degrading carbohydrate active enzymes (CAZymes). These data boost genome sequence representation across the Phytophthora genus, and form resources for further study of Phytophthora pathogenesis.

Secreted pectin monooxygenases drive plant infection by pathogenic oomycetes.

The oomycete Phytophthora infestans is a damaging crop pathogen and a model organism to study plant-pathogen interactions. We report the discovery of a family of copper-dependent lytic polysaccharide monooxygenases (LPMOs) in plant pathogenic oomycetes and its role in plant infection by P. infestans We show that LPMO-encoding genes are up-regulated early during infection and that the secreted enzymes oxidatively cleave the backbone of pectin, a charged polysaccharide in the plant cell wall. The crystal structure of the most abundant of these LPMOs sheds light on its ability to recognize and degrade pectin, and silencing the encoding gene in P. infestans inhibits infection of potato, indicating a role in host penetration. The identification of LPMOs as virulence factors in pathogenic oomycetes opens up opportunities in crop protection and food security.

Haustorium formation and a distinct biotrophic transcriptome characterize infection of Nicotiana benthamiana by the tree pathogen Phytophthora kernoviae.

Phytophthora species cause some of the most serious diseases of trees and threaten forests in many parts of the world. Despite the generation of genome sequence assemblies for over 10 tree-pathogenic Phytophthora species and improved detection methods, there are many gaps in our knowledge of how these pathogens interact with their hosts. To facilitate cell biology studies of the infection cycle we examined whether the tree pathogen Phytophthora kernoviae could infect the model plant Nicotiana benthamiana. We transformed P. kernoviae to express green fluorescent protein (GFP) and demonstrated that it forms haustoria within infected N. benthamiana cells. Haustoria were also formed in infected cells of natural hosts, Rhododendron ponticum and European beech (Fagus sylvatica). We analysed the transcriptome of P. kernoviae in cultured mycelia, spores, and during infection of N. benthamiana, and detected 12,559 transcripts. Of these, 1,052 were predicted to encode secreted proteins, some of which may function as effectors to facilitate disease development. From these, we identified 87 expressed candidate RXLR (Arg-any amino acid-Leu-Arg) effectors. We transiently expressed 12 of these as GFP fusions in N. benthamiana leaves and demonstrated that nine significantly enhanced P. kernoviae disease progression and diversely localized to the cytoplasm, nucleus, nucleolus, and plasma membrane. Our results show that N. benthamiana can be used as a model host plant for studying this tree pathogen, and that the interaction likely involves suppression of host immune responses by RXLR effectors. These results establish a platform to expand the understanding of Phytophthora tree diseases.