High-efficiency green management of potato late blight by a self-assembled multicomponent nano-bioprotectant.

Potato late blight caused by Phytophthora infestans is a devastating disease worldwide. Unlike other plant pathogens, double-stranded RNA (dsRNA) is poorly taken up by P. infestans, which is a key obstacle in using dsRNA for disease control. Here, a self-assembled multicomponent nano-bioprotectant for potato late blight management is designed based on dsRNA and a plant elicitor. Nanotechnology overcomes the dsRNA delivery bottleneck for P. infestans and extends the RNAi protective window. The protective effect of nano-enabled dsRNA against infection arises from a synergistic mechanism that bolsters the stability of dsRNA and optimizes its effective intracellular delivery. Additionally, the nano-enabled elicitor enhances endocytosis and amplifies the systemic defense response of the plants. Co-delivery of dsRNA and an elicitor provides a protective effect via the two aspects of pathogen inhibition and elevated plant defense mechanisms. The multicomponent nano-bioprotectant exhibits superior control efficacy compared to a commercial synthetic pesticide in field conditions. This work proposes an eco-friendly strategy to manage devastating plant diseases and pests.

Comparative transcriptome profiling provides insights into the growth promotion activity of Pseudomonas fluorescens strain SLU99 in tomato and potato plants.

The use of biocontrol agents with plant growth-promoting activity has emerged as an approach to support sustainable agriculture. During our field evaluation of potato plants treated with biocontrol rhizobacteria, four bacteria were associated with increased plant height. Using two important solanaceous crop plants, tomato and potato, we carried out a comparative analysis of the growth-promoting activity of the four bacterial strains: Pseudomonas fluorescens SLU99, Serratia plymuthica S412, S. rubidaea AV10, and S. rubidaea EV23. Greenhouse and in vitro experiments showed that P. fluorescens SLU99 promoted plant height, biomass accumulation, and yield of potato and tomato plants, while EV23 promoted growth in potato but not in tomato plants. SLU99 induced the expression of plant hormone-related genes in potato and tomato, especially those involved in maintaining homeostasis of auxin, cytokinin, gibberellic acid and ethylene. Our results reveal potential mechanisms underlying the growth promotion and biocontrol effects of these rhizobacteria and suggest which strains may be best deployed for sustainably improving crop yield.

RNA silencing proteins and small RNAs in oomycete plant pathogens and biocontrol agents.

Introduction: Oomycetes cause several damaging diseases of plants and animals, and some species also act as biocontrol agents on insects, fungi, and other oomycetes. RNA silencing is increasingly being shown to play a role in the pathogenicity of Phytophthora species, either through trans-boundary movement of small RNAs (sRNAs) or through expression regulation of infection promoting effectors. Methods: To gain a wider understanding of RNA silencing in oomycete species with more diverse hosts, we mined genome assemblies for Dicer-like (DCL), Argonaute (AGO), and RNA dependent RNA polymerase (RDRP) proteins from Phytophthora plurivora, Ph. cactorum, Ph. colocasiae, Pythium oligandrum, Py. periplocum, and Lagenidium giganteum. Moreover, we sequenced small RNAs from the mycelium stage in each of these species. Results and discussion: Each of the species possessed a single DCL protein, but they differed in the number and sequence of AGOs and RDRPs. SRNAs of 21nt, 25nt, and 26nt were prevalent in all oomycetes analyzed, but the relative abundance and 5' base preference of these classes differed markedly between genera. Most sRNAs mapped to transposons and other repeats, signifying that the major role for RNA silencing in oomycetes is to limit the expansion of these elements. We also found that sRNAs may act to regulate the expression of duplicated genes. Other sRNAs mapped to several gene families, and this number was higher in Pythium spp., suggesting a role of RNA silencing in regulating gene expression. Genes for most effector classes were the source of sRNAs of variable size, but some gene families showed a preference for specific classes of sRNAs, such as 25/26 nt sRNAs targeting RxLR effector genes in Phytophthora species. Novel miRNA-like RNAs (milRNAs) were discovered in all species, and two were predicted to target transcripts for RxLR effectors in Ph. plurivora and Ph. cactorum, indicating a putative role in regulating infection. Moreover, milRNAs from the biocontrol Pythium species had matches in the predicted transcriptome of Phytophthora infestans and Botrytis cinerea, and L. giganteum milRNAs matched candidate genes in the mosquito Aedes aegypti. This suggests that trans-boundary RNA silencing may have a role in the biocontrol action of these oomycetes.

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.

Antagonistic and plant growth promotion of rhizobacteria against Phytophthora colocasiae in taro.

Taro leaf blight caused by Phytophthora colocasiae adversely affects the growth and yield of taro. The management of this disease depends heavily on synthetic fungicides. These compounds, however, pose potential hazards to human health and the environment. The present study aimed to investigate an alternative approach for plant growth promotion and disease control by evaluating seven different bacterial strains (viz., Serratia plymuthica, S412; S. plymuthica, S414; S. plymuthica, AS13; S. proteamaculans, S4; S. rubidaea, EV23; S. rubidaea, AV10; Pseudomonas fluorescens, SLU-99) and their different combinations as consortia against P. colocasiae. Antagonistic tests were performed in in vitro plate assays and the effective strains were selected for detached leaf assays and greenhouse trials. Plant growth-promoting and disease prevention traits of selected bacterial strains were also investigated in vitro. Our results indicated that some of these strains used singly (AV10, AS13, S4, and S414) and in combinations (S4+S414, AS13+AV10) reduced the growth of P. colocasiae (30-50%) in vitro and showed disease reduction ability when used singly or in combinations as consortia in greenhouse trials (88.75-99.37%). The disease-suppressing ability of these strains may be related to the production of enzymes such as chitinase, protease, cellulase, and amylase. Furthermore, all strains tested possessed plant growth-promoting traits such as indole-3-acetic acid production, siderophore formation, and phosphate solubilization. Overall, the present study revealed that bacterial strains significantly suppressed P. colocasiae disease development using in vitro, detached leaf, and greenhouse assays. Therefore, these bacterial strains can be used as an alternative strategy to minimize the use of synthetic fungicides and fertilizers to control taro blight and improve sustainable taro production.

Spray-Induced Gene Silencing to Study Gene Function in Phytophthora.

RNA interference (RNAi) is a conserved cellular defense mechanism mediated by double-stranded RNA (dsRNA) that can regulate gene expression through targeted destruction of mRNAs (messenger RNAs). Recent studies have shown that spraying dsRNAs or small RNAs (sRNAs) that target essential genes of pathogens on plant surfaces can confer protection against pests and pathogens. Also called spray-induced gene silencing (SIGS), this strategy can be used for disease control and for transient gene silencing to study the function of genes in plant-pathogen interactions. Furthermore, as sRNAs can move locally, systemically, and cross-kingdom during plant-microbe interactions, SIGS allows quick detection and characterization of gene functions in pathogens and plants.

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.

Spray-Induced Gene Silencing as a Potential Tool to Control Potato Late Blight Disease.

Phytophthora infestans causes late blight disease on potato and tomato and is currently controlled by resistant cultivars or intensive fungicide spraying. Here, we investigated an alternative means for late blight control by spraying potato leaves with double-stranded RNAs (dsRNA) that target the P. infestans genes essential for infection. First, we showed that the sporangia of P. infestans expressing green fluorescent protein (GFP) can take up in vitro synthesized dsRNAs homologous to GFP directly from their surroundings, including leaves, which led to the reduced relative expression of GFP. We further demonstrate the potential of spray-induced gene silencing (SIGS) in controlling potato late blight disease by targeting developmentally important genes in P. infestans such as guanine-nucleotide binding protein ß-subunit (PiGPB1), haustorial membrane protein (PiHmp1), cutinase (PiCut3), and endo-1,3(4)-ß-glucanase (PiEndo3). Our results demonstrate that SIGS can potentially be used to mitigate potato late blight; however, the degree of disease control is dependent on the selection of the target genes.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

Devastating intimacy: the cell biology of plant-Phytophthora interactions.

An understanding of the cell biology underlying the burgeoning molecular genetic and genomic knowledge of oomycete pathogenicity is essential to gain the full context of how these pathogens cause disease on plants. An intense research focus on secreted Phytophthora effector proteins, especially those containing a conserved N-terminal RXLR motif, has meant that most cell biological studies into Phytophthora diseases have focussed on the effectors and their host target proteins. While these effector studies have provided novel insights into effector secretion and host defence mechanisms, there remain many unanswered questions about fundamental processes involved in spore biology, host penetration and haustorium formation and function.

Phytophthora infestans RXLR effectors act in concert at diverse subcellular locations to enhance host colonization.

Oomycetes such as the potato blight pathogen Phytophthora infestans deliver RXLR effectors into plant cells to manipulate host processes and promote disease. Knowledge of where they localize inside host cells is important in understanding their function. Fifty-two P. infestans RXLR effectors (PiRXLRs) up-regulated during early stages of infection were expressed as fluorescent protein (FP) fusions inside cells of the model host Nicotiana benthamiana. FP-PiRXLR fusions were predominantly nucleo-cytoplasmic, nuclear, or plasma membrane-associated. Some also localized to the endoplasmic reticulum, mitochondria, peroxisomes, or microtubules, suggesting diverse sites of subcellular activity. Seven of the 25 PiRXLRs examined during infection accumulated at sites of haustorium penetration, probably due to co-localization with host target processes; Pi16663 (Avr1), for example, localized to Sec5-associated mobile bodies which showed perihaustorial accumulation. Forty-five FP-RXLR fusions enhanced pathogen leaf colonization when expressed in Nicotiana benthamiana, revealing that their presence was beneficial to infection. Co-expression of PiRXLRs that target and suppress different immune pathways resulted in an additive enhancement of colonization, indicating the potential to study effector combinations using transient expression assays. We provide a broad platform of high confidence P. infestans effector candidates from which to investigate the mechanisms, singly and in combination, by which this pathogen causes disease.

The Phytophthora infestans Haustorium Is a Site for Secretion of Diverse Classes of Infection-Associated Proteins.

The oomycete potato blight pathogen Phytophthora infestans secretes a diverse set of proteins to manipulate host plant immunity. However, there is limited knowledge about how and where they are secreted during infection. Here we used the endoplasmic reticulum (ER)-to-Golgi secretion pathway inhibitor brefeldin A (BFA) in combination with liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) to identify extracellular proteins from P. infestans that were conventionally secreted from in vitro-cultured hyphae. We identified 19 proteins with predicted signal peptides that potentially influence plant interactions for which secretion was attenuated by BFA. In addition to inhibition by the apoplastic effector EPIC1, a cysteine protease inhibitor, we show that secretion of the cell wall-degrading pectinesterase enzyme PE1 and the microbe-associated molecular pattern (MAMP)-like elicitin INF4 was inhibited by BFA in vitro and in planta, demonstrating that these proteins are secreted by the conventional, Golgi-mediated pathway. For comparison, secretion of a cytoplasmic RXLR (Arg-[any amino acid]-Leu-Arg) effector, Pi22926, was not inhibited by BFA. During infection, whereas INF4 accumulated outside the plant cell, RXLR effector Pi22926 entered the plant cell and accumulated in the nucleus. The P. infestans effectors, the PE1 enzyme, and INF4 were all secreted from haustoria, pathogen structures that penetrate the plant cell wall to form an intimate interaction with the host plasma membrane. Our findings show the haustorium to be a major site of both conventional and nonconventional secretion of proteins with diverse functions during infection.IMPORTANCE There are many different classes of proteins secreted from Phytophthora infestans that may influence or facilitate infection. Elucidating where and how they are secreted during infection is an important step toward developing methods to control their delivery processes. We used an inhibitor of conventional secretion to identify the following different classes of infection-associated extracellular proteins: cell wall-degrading and cell wall-modifying enzymes, microbe-associated molecular pattern-like proteins that may elicit immune responses, and apoplastic effectors that are predicted to suppress immunity. In contrast, secretion of a cytoplasmic effector that is translocated into host cells is nonconventional, as it is insensitive to inhibitor treatment. This evidence further supports the finding that proteins that are active in the apoplast and effector proteins that are active in the host cytoplasm are differentially secreted by P. infestans Critically, it demonstrates that a disease-specific developmental structure, the haustorium, is a major secretion site for diverse protein classes during infection.

Draft Genome Sequence for the Tree Pathogen Phytophthora plurivora.

Species from the genus Phytophthora are well represented among organisms causing serious diseases on trees. Phytophthora plurivora has been implicated in long-term decline of woodland trees across Europe. Here we present a draft genome sequence of P. plurivora, originally isolated from diseased European beech (Fagus sylvatica) in Malmö, Sweden. When compared with other sequenced Phytophthora species, the P. plurivora genome assembly is relatively compact, spanning 41 Mb. This is organized in 1,919 contigs and 1,898 scaffolds, encompassing 11,741 predicted genes, and has a repeat content of approximately 15%. Comparison of allele frequencies revealed evidence for tetraploidy in the sequenced isolate. As in other sequenced Phytophthora species, P. plurivora possesses genes for pathogenicity-associated RXLR and Crinkle and Necrosis effectors, predominantly located in gene-sparse genomic regions. Comparison of the P. plurivora RXLR effectors with orthologs in other sequenced species in the same clade (Phytophthora multivora and Phytophthora capsici) revealed that the orthologs were likely to be under neutral or purifying selection.

Genome Sequence Resource for the Oomycete Taro Pathogen Phytophthora colocasiae.

Phytophthora colocasiae is a phytopathogenic oomycete that causes leaf blight and corm rot on taro (Colocasia esculenta), an important staple crop in the tropics. The impact of P. colocasiae is a serious concern for food security in Asian and Oceanic regions. Vietnamese strain 7290 of P. colocasiae was sequenced (Illumina) to assemble a draft genome of 56.6 Mb, comprised of 19,853 scaffolds and 19,984 predicted protein-coding genes. As in other Phytophthora species, P. colocasiae possesses numerous pathogenicity-related genes, such as the RxLR class of effectors. This draft genome sequence of P. colocasiae provides a resource to underpin the first steps in determining the molecular mechanisms of disease development in this pathosystem.

Delivery of cytoplasmic and apoplastic effectors from Phytophthora infestans haustoria by distinct secretion pathways.

The potato blight pathogen Phytophthora infestans secretes effector proteins that are delivered inside (cytoplasmic) or can act outside (apoplastic) plant cells to neutralize host immunity. Little is known about how and where effectors are secreted during infection, yet such knowledge is essential to understand and combat crop disease. We used transient Agrobacterium tumefaciens-mediated in planta expression, transformation of P. infestans with fluorescent protein fusions and confocal microscopy to investigate delivery of effectors to plant cells during infection. The cytoplasmic effector Pi04314, expressed as a monomeric red fluorescent protein (mRFP) fusion protein with a signal peptide to secrete it from plant cells, did not passively re-enter the cells upon secretion. However, Pi04314-mRFP expressed in P. infestans was translocated from haustoria, which form intimate interactions with plant cells, to accumulate at its sites of action in the host nucleus. The well-characterized apoplastic effector EPIC1, a cysteine protease inhibitor, was also secreted from haustoria. EPIC1 secretion was inhibited by brefeldin A (BFA), demonstrating that it is delivered by conventional Golgi-mediated secretion. By contrast, Pi04314 secretion was insensitive to BFA treatment, indicating that the cytoplasmic effector follows an alternative route for delivery into plant cells. Phytophthora infestans haustoria are thus sites for delivery of both apoplastic and cytoplasmic effectors during infection, following distinct secretion pathways.

Draft genome of the oomycete pathogen Phytophthora cactorum strain LV007 isolated from European beech (Fagus sylvatica).

Phytophthora cactorum is a broad host range phytopathogenic oomycete. P. cactorum strain LV007 was isolated from a diseased European Beech (Fagus sylvatica) in Malmö, Sweden in 2016. The draft genome of P. cactorum strain LV007 is 67.81 Mb. It contains 15,567 contigs and 21,876 predicted protein-coding genes. As reported for other phytopathogenic Phytophthora species, cytoplasmic effector proteins including RxLR and CRN families were identified. The genome sequence has been deposited at DDBJ/ENA/GenBank under the accession NBIJ00000000. The version described in this paper is version NBIJ01000000.

The cell biology of late blight disease.

Late blight, caused by the oomycete Phytophthora infestans, is a major global disease of potato and tomato. Cell biology is teaching us much about the developmental stages associated with infection, especially the haustorium, which is a site of intimate interaction and molecular exchange between pathogen and host. Recent observations suggest a role for the plant endocytic cycle in specific recruitment of host proteins to the Extra-Haustorial Membrane, emphasising the unique nature of this membrane compartment. In addition, there has been a strong focus on the activities of RXLR effectors, which are delivered into plant cells to modulate and manipulate host processes. RXLR effectors interact directly with diverse plant proteins at a range of subcellular locations to promote disease.

Fragmentation of tRNA in Phytophthora infestans asexual life cycle stages and during host plant infection.

BACKGROUND: The oomycete Phytophthora infestans possesses active RNA silencing pathways, which presumably enable this plant pathogen to control the large numbers of transposable elements present in its 240 Mb genome. Small RNAs (sRNAs), central molecules in RNA silencing, are known to also play key roles in this organism, notably in regulation of critical effector genes needed for infection of its potato host. RESULTS: To identify additional classes of sRNAs in oomycetes, we mapped deep sequencing reads to transfer RNAs (tRNAs) thereby revealing the presence of 19-40 nt tRNA-derived RNA fragments (tRFs). Northern blot analysis identified abundant tRFs corresponding to half tRNA molecules. Some tRFs accumulated differentially during infection, as seen by examining sRNAs sequenced from P. infestans-potato interaction libraries. The putative connection between tRF biogenesis and the canonical RNA silencing pathways was investigated by employing hairpin RNA-mediated RNAi to silence the genes encoding P. infestans Argonaute (PiAgo) and Dicer (PiDcl) endoribonucleases. By sRNA sequencing we show that tRF accumulation is PiDcl1-independent, while Northern hybridizations detected reduced levels of specific tRNA-derived species in the PiAgo1 knockdown line. CONCLUSIONS: Our findings extend the sRNA diversity in oomycetes to include fragments derived from non-protein-coding RNA transcripts and identify tRFs with elevated levels during infection of potato by P. infestans.

Sequence diversity in the large subunit of RNA polymerase I contributes to Mefenoxam insensitivity in Phytophthora infestans.

Phenylamide fungicides have been widely used for the control of oomycete-incited plant diseases for over 30 years. Insensitivity to this chemical class of fungicide was recorded early in its usage history, but the precise protein(s) conditioning insensitivity has proven difficult to determine. To determine the genetic basis of insensitivity and to inform strategies for the cloning of the gene(s) responsible, genetic crosses were established between Mefenoxam sensitive and intermediate insensitive isolates of Phytophthora infestans, the potato late blight pathogen. F1 progeny showed the expected semi-dominant phenotypes for Mefenoxam insensitivity and suggested the involvement of multiple loci, complicating the positional cloning of the gene(s) conditioning insensitivity to Mefenoxam. Instead, a candidate gene strategy was used, based on previous observations that the primary effect of phenylamide compounds is to inhibit ribosomal RNA synthesis. The subunits of RNA polymerase I (RNApolI) were sequenced from sensitive and insensitive isolates and F1 progeny. Single nucleotide polymorphisms (SNPs) specific to insensitive field isolates were identified in the gene encoding the large subunit of RNApolI. In a survey of field isolates, SNP T1145A (Y382F) showed an 86% association with Mefenoxam insensitivity. Isolates not showing this association belonged predominantly to one P. infestans genotype. The transfer of the 'insensitive' allele of RPA190 to a sensitive isolate yielded transgenic lines that were insensitive to Mefenoxam. These results demonstrate that sequence variation in RPA190 contributes to insensitivity to Mefenoxam in P. infestans.