Functional diversification of a wild potato immune receptor at its center of origin.

Plant cell surface pattern recognition receptors (PRRs) and intracellular immune receptors cooperate to provide immunity to microbial infection. Both receptor families have coevolved at an accelerated rate, but the evolution and diversification of PRRs is poorly understood. We have isolated potato surface receptor Pep-13 receptor unit (PERU) that senses Pep-13, a conserved immunogenic peptide pattern from plant pathogenic Phytophthora species. PERU, a leucine-rich repeat receptor kinase, is a bona fide PRR that binds Pep-13 and enhances immunity to Phytophthora infestans infection. Diversification in ligand binding specificities of PERU can be traced to sympatric wild tuber-bearing Solanum populations in the Central Andes. Our study reveals the evolution of cell surface immune receptor alleles in wild potato populations that recognize ligand variants not recognized by others.

Plant cell surface immune receptors-Novel insights into function and evolution.

Plants use surface resident and intracellular immune receptors to provide robust immunity against microbial infections. The contribution of the two receptor types to plant immunity differs spatially and temporally. The ongoing identification of new plant cell surface immune receptors and their microbial-derived immunogenic ligands reveal a previously unexpected complexity of plant surface sensors involved in the detection of specific microbial species. Comparative analyses of the plant species distribution of cell surface immune receptors indicate that plants harbor larger sets of genus- or species-specific surface receptors in addition to very few widespread pattern sensors. Leucine-rich repeat surface and intracellular immune sensors emerge as two polymorphic receptor classes whose evolutionary trajectories appear to be linked. This is consistent with their functional cooperativity in providing full plant immunity.

The Arabidopsis TIR-NBS-LRR protein CSA1 guards BAK1-BIR3 homeostasis and mediates convergence of pattern- and effector-induced immune responses.

Arabidopsis BAK1/SERK3, a co-receptor of leucine-rich repeat pattern recognition receptors (PRRs), mediates pattern-triggered immunity (PTI). Genetic inactivation of BAK1 or BAK1-interacting receptor-like kinases (BIRs) causes cell death, but the direct mechanisms leading to such deregulation remains unclear. Here, we found that the TIR-NBS-LRR protein CONSTITUTIVE SHADE AVOIDANCE 1 (CSA1) physically interacts with BIR3, but not with BAK1. CSA1 mediates cell death in bak1-4 and bak1-4 bir3-2 mutants via components of effector-triggered immunity-(ETI) pathways. Effector HopB1-mediated perturbation of BAK1 also results in CSA1-dependent cell death. Likewise, microbial pattern pg23-induced cell death, but not PTI responses, requires CSA1. Thus, we show that CSA1 guards BIR3 BAK1 homeostasis and integrates pattern- and effector-mediated cell death pathways downstream of BAK1. De-repression of CSA1 in the absence of intact BAK1 and BIR3 triggers ETI cell death. This suggests that PTI and ETI pathways are activated downstream of BAK1 for efficient plant immunity.

Genotyping-by-sequencing-based identification of Arabidopsis pattern recognition receptor RLP32 recognizing proteobacterial translation initiation factor IF1.

Activation of plant pattern-triggered immunity (PTI) relies on the recognition of microbe-derived structures, termed patterns, through plant-encoded surface-resident pattern recognition receptors (PRRs). We show that proteobacterial translation initiation factor 1 (IF1) triggers PTI in Arabidopsis thaliana and related Brassicaceae species. Unlike for most other immunogenic patterns, IF1 elicitor activity cannot be assigned to a small peptide epitope, suggesting that tertiary fold features are required for IF1 receptor activation. We have deployed natural variation in IF1 sensitivity to identify Arabidopsis leucine-rich repeat (LRR) receptor-like protein 32 (RLP32) as IF1 receptor using a restriction site-associated DNA sequencing approach. RLP32 confers IF1 sensitivity to rlp32 mutants, IF1-insensitive Arabidopsis accessions and IF1-insensitive Nicotiana benthamiana, binds IF1 specifically and forms complexes with LRR receptor kinases SOBIR1 and BAK1 to mediate signaling. Similar to other PRRs, RLP32 confers resistance to Pseudomonas syringae, highlighting an unexpectedly complex array of bacterial pattern sensors within a single plant species.

The EDS1-PAD4-ADR1 node mediates Arabidopsis pattern-triggered immunity.

Plants deploy cell-surface and intracellular leucine rich-repeat domain (LRR) immune receptors to detect pathogens1. LRR receptor kinases and LRR receptor proteins at the plasma membrane recognize microorganism-derived molecules to elicit pattern-triggered immunity (PTI), whereas nucleotide-binding LRR proteins detect microbial effectors inside cells to confer effector-triggered immunity (ETI). Although PTI and ETI are initiated in different host cell compartments, they rely on the transcriptional activation of similar sets of genes2, suggesting pathway convergence upstream of nuclear events. Here we report that PTI triggered by the Arabidopsis LRR receptor protein RLP23 requires signalling-competent dimers of the lipase-like proteins EDS1 and PAD4, and of ADR1 family helper nucleotide-binding LRRs, which are all components of ETI. The cell-surface LRR receptor kinase SOBIR1 links RLP23 with EDS1, PAD4 and ADR1 proteins, suggesting the formation of supramolecular complexes containing PTI receptors and transducers at the inner side of the plasma membrane. We detected similar evolutionary patterns in LRR receptor protein and nucleotide-binding LRR genes across Arabidopsis accessions; overall higher levels of variation in LRR receptor proteins than in LRR receptor kinases are consistent with distinct roles of these two receptor families in plant immunity. We propose that the EDS1-PAD4-ADR1 node is a convergence point for defence signalling cascades, activated by both surface-resident and intracellular LRR receptors, in conferring pathogen immunity.

Distinct immune sensor systems for fungal endopolygalacturonases in closely related Brassicaceae.

Plant pattern recognition receptors (PRRs) facilitate recognition of microbial patterns and mediate activation of plant immunity. Arabidopsis thaliana RLP42 senses fungal endopolygalacturonases (PGs) and triggers plant defence through complex formation with SOBIR1 and SERK co-receptors. Here, we show that a conserved 9-amino-acid fragment pg9(At) within PGs is sufficient to activate RLP42-dependent plant immunity. Structure-function analysis reveals essential roles of amino acid residues within the RLP42 leucine-rich repeat and island domains for ligand binding and PRR complex assembly. Sensitivity to pg9(At), which is restricted to A. thaliana and exhibits scattered accession specificity, is unusual for known PRRs. Arabidopsis arenosa and Brassica rapa, two Brassicaceae species closely related to A. thaliana, respectively perceive immunogenic PG fragments pg20(Aa) and pg36(Bra), which are structurally distinct from pg9(At). Our study provides evidence for rapid evolution of polymorphic PG sensors with distinct pattern specificities within a single plant family.

Phytophthora infestans small phospholipase D-like proteins elicit plant cell death and promote virulence.

The successful invasion of host tissue by (hemi-)biotrophic plant pathogens is dependent on modifications of the host plasma membrane to facilitate the two-way transfer of proteins and other compounds. Haustorium formation and the establishment of extrahaustorial membranes are probably dependent on a variety of enzymes that modify membranes in a coordinated fashion. Phospholipases, enzymes that hydrolyse phospholipids, have been implicated as virulence factors in several pathogens. The oomycete Phytophthora infestans is a hemibiotrophic pathogen that causes potato late blight. It possesses different classes of phospholipase D (PLD) proteins, including small PLD-like proteins with and without signal peptide (sPLD-likes and PLD-likes, respectively). Here, we studied the role of sPLD-like-1, sPLD-like-12 and PLD-like-1 in the infection process. They are expressed in expanding lesions on potato leaves and during in vitro growth, with the highest transcript levels in germinating cysts. When expressed in planta in the presence of the silencing suppressor P19, all three elicited a local cell death response that was visible at the microscopic level as autofluorescence and strongly boosted in the presence of calcium. Moreover, inoculation of leaves expressing the small PLD-like genes resulted in increased lesion growth and greater numbers of sporangia, but this was abolished when mutated PLD-like genes were expressed with non-functional PLD catalytic motifs. These results show that the three small PLD-likes are catalytically active and suggest that their enzymatic activity is required for the promotion of virulence, possibly by executing membrane modifications to support the growth of P. infestans in the host.

Phytophthora infestans RXLR effector SFI5 requires association with calmodulin for PTI/MTI suppressing activity.

Pathogens secrete effector proteins to interfere with plant innate immunity, in which Ca2+ /calmodulin (CaM) signalling plays key roles. Thus far, few effectors have been identified that directly interact with CaM for defence suppression. Here, we report that SFI5, an RXLR effector from Phytophthora infestans, suppresses microbe-associated molecular pattern (MAMP)-triggered immunity (MTI) by interacting with host CaMs. We predicted the CaM-binding site in SFI5 using in silico analysis. The interaction between SFI5 and CaM was tested by both in vitro and in vivo assays. MTI suppression by SFI5 and truncated variants were performed in a tomato protoplast system. We found that both the predicted CaM-binding site and the full-length SFI5 protein interact with CaM in the presence of Ca2+ . MTI responses, such as FRK1 upregulation, reactive oxygen species accumulation, and mitogen-activated protein kinase activation were suppressed by truncated SFI5 proteins containing the C-terminal CaM-binding site but not by those without it. The plasma membrane localization of SFI5 and its ability to enhance infection were also perturbed by loss of the CaM-binding site. We conclude that CaM-binding is required for localization and activity of SFI5. We propose that SFI5 suppresses plant immunity by interfering with immune signalling components after activation by CaMs.

Trans-Kingdom RNA Silencing in Plant-Fungal Pathogen Interactions.

Fungal pathogens represent a major group of plant invaders that are the causative agents of many notorious plant diseases. Large quantities of RNAs, especially small RNAs involved in gene silencing, have been found to transmit bidirectionally between fungal pathogens and their hosts. Although host-induced gene silencing (HIGS) technology has been developed and applied to protect crops from fungal infections, the mechanisms of RNA transmission, especially small RNAs regulating trans-kingdom RNA silencing in plant immunity, are largely unknown. In this review, we summarize and discuss recent important findings regarding trans-kingdom sRNAs and RNA silencing in plant-fungal pathogen interactions compared with the well-known RNAi mechanisms in plants and fungi. We focus on the interactions between plant and fungal pathogens with broad hosts, represented by the vascular pathogen Verticillium dahliae and non-vascular pathogen Botrytis cinerea, and discuss the known instances of natural RNAi transmission between fungal pathogens and host plants. Given that HIGS has been developed and recently applied in controlling Verticillium wilt diseases, we propose an ideal research system exploiting plant vasculature-Verticillium interaction to further study trans-kingdom RNA silencing.

Expression of pathogenesis-related genes in cotton roots in response to Verticillium dahliae PAMP molecules.

Verticillium wilt disease becomes a major threat to many economically important crops. It is unclear whether and how plant immunity takes place during cotton-Verticillium interaction due to the lack of marker genes. Taking advantage of cotton (Gossypium hirsutum) genome, we discovered pathogenesis-related (PR) gene families, which have been widely used as markers of immune responses in plants. To profile the expression of G. hirsutum PR genes in the process of plant immunity, we treated cotton roots with two immunogenic peptides, flg22 and nlp20 known as pathogen-associated molecular patterns, as well as three Verticillium dahliae-derived peptides, nlp20Vd2, nlp23Vd3, and nlp23Vd4 which are highly identical to nlp20. Quantitative real-time PCR results revealed that 14 G. hirsutum PR gene (GhPR) families were induced or suppressed independently in response to flg22, nlp20, nlp20Vd2, nlp23Vd3, and nlp23Vd4. Most GhPR genes are expressed highest at 3 h post incubation of immunogenic peptides. Compared to flg22 and nlp20, nlp20Vd2 is more effective to trigger up-regulated expression of GhPR genes. Notably, both nlp23Vd3 and nlp23Vd4 are able to induce GhPR gene up-regulation, although they do not induce necrosis on cotton leaves. Thus, our results provide marker genes and new immunogenic peptides for further investigation of cotton-V. dahliae interaction.

VdPKS1 is required for melanin formation and virulence in a cotton wilt pathogen Verticillium dahliae.

Verticillium dahliae is a soil-borne phytopathogenic fungus that causes vascular wilt disease in a broad range of hosts. This pathogen survives for many years in soil in the form of melanized microsclerotia. To investigate the melanin synthesis in V. dahliae, we identified a polyketide synthase gene in V. dahliae, namely VdPKS1. PKS1 is known to involve in the dihydroxynaphthalene melanin synthesis pathway in many fungi. We found that VdPKS1 was required for melanin formation but not for microsclerotial production in V. dahliae. The VdPKS1 gene-disruption mutant (vdpks1) formed melanin-deficient albino microsclerotia, which did not affect the fungal colonization in host tissues but significantly reduced the disease severity. Gene transcription analysis in the wild-type and the vdpks1 strains suggested that VdPKS1 gene-disruption influenced the expression of a series of genes involved in ethylene biosynthesis, microsclerotial formation and pathogenesis. Our results suggest that the VdPKS1-mediated melanin synthesis is important for virulence and developmental traits of V. dahliae.

The Verticillium-specific protein VdSCP7 localizes to the plant nucleus and modulates immunity to fungal infections.

Fungal pathogens secrete effector proteins to suppress plant basal defense for successful colonization. Resistant plants, however, can recognize effectors by cognate R proteins to induce effector-triggered immunity (ETI). By analyzing secretomes of the vascular fungal pathogen Verticillium dahliae, we identified a novel secreted protein VdSCP7 that targets the plant nucleus. The green fluorescent protein (GFP)-tagged VdSCP7 gene with either a mutated nuclear localization signal motif or with additional nuclear export signal was transiently expressed in Nicotiana benthamiana, and investigated for induction of plant immunity. The role of VdSCP7 in V. dahliae pathogenicity was characterized by gene knockout and complementation, and GFP labeling. Expression of the VdSCP7 gene in N. benthamiana activated both salicylic acid and jasmonate signaling, and altered the plant's susceptibility to the pathogens Botrytis cinerea and Phytophthora capsici. The immune response activated by VdSCP7 was highly dependent on its initial extracellular secretion and subsequent nuclear localization in plants. Knockout of the VdSCP7 gene significantly enhanced V. dahliae aggressiveness on cotton. GFP-labeled VdSCP7 is secreted by V. dahliae and accumulates in the plant nucleus. We conclude that VdSCP7 is a novel effector protein that targets the host nucleus to modulate plant immunity, and suggest that plants can recognize VdSCP7 to activate ETI during fungal infection.

Cotton plants export microRNAs to inhibit virulence gene expression in a fungal pathogen.

Plant pathogenic fungi represent the largest group of disease-causing agents on crop plants, and are a constant and major threat to agriculture worldwide. Recent studies have shown that engineered production of RNA interference (RNAi)-inducing dsRNA in host plants can trigger specific fungal gene silencing and confer resistance to fungal pathogens1-7. Although these findings illustrate efficient uptake of host RNAi triggers by pathogenic fungi, it is unknown whether or not such an uptake mechanism has been evolved for a natural biological function in fungus-host interactions. Here, we show that in response to infection with Verticillium dahliae (a vascular fungal pathogen responsible for devastating wilt diseases in many crops) cotton plants increase production of microRNA 166 (miR166) and miR159 and export both to the fungal hyphae for specific silencing. We found that two V. dahliae genes encoding a Ca2+-dependent cysteine protease (Clp-1) and an isotrichodermin C-15 hydroxylase (HiC-15), and targeted by miR166 and miR159, respectively, are both essential for fungal virulence. Notably, V. dahliae strains expressing either Clp-1 or HiC-15 rendered resistant to the respective miRNA exhibited drastically enhanced virulence in cotton plants. Together, our findings identify a novel defence strategy of host plants by exporting specific miRNAs to induce cross-kingdom gene silencing in pathogenic fungi and confer disease resistance.

An Improved Single-Step Cloning Strategy Simplifies the Agrobacterium tumefaciens-Mediated Transformation (ATMT)-Based Gene-Disruption Method for Verticillium dahliae.

The soilborne fungal pathogen Verticillium dahliae infects a broad range of plant species to cause severe diseases. The availability of Verticillium genome sequences has provided opportunities for large-scale investigations of individual gene function in Verticillium strains using Agrobacterium tumefaciens-mediated transformation (ATMT)-based gene-disruption strategies. Traditional ATMT vectors require multiple cloning steps and elaborate characterization procedures to achieve successful gene replacement; thus, these vectors are not suitable for high-throughput ATMT-based gene deletion. Several advancements have been made that either involve simplification of the steps required for gene-deletion vector construction or increase the efficiency of the technique for rapid recombinant characterization. However, an ATMT binary vector that is both simple and efficient is still lacking. Here, we generated a USER-ATMT dual-selection (DS) binary vector, which combines both the advantages of the USER single-step cloning technique and the efficiency of the herpes simplex virus thymidine kinase negative-selection marker. Highly efficient deletion of three different genes in V. dahliae using the USER-ATMT-DS vector enabled verification that this newly-generated vector not only facilitates the cloning process but also simplifies the subsequent identification of fungal homologous recombinants. The results suggest that the USER-ATMT-DS vector is applicable for efficient gene deletion and suitable for large-scale gene deletion in V. dahliae.

The dual edge of RNA silencing suppressors in the virus-host interactions.

RNA silencing (or RNA interference, RNAi) plays a key role in the plant antiviral defense. To facilitate infection, viruses encode suppressors of RNA silencing (VSRs) to counteract antiviral defense. In the co-evolutionary arms race between hosts and viruses, extreme viral accumulation does not benefit either hosts or viruses. During viral infection, antiviral silencing and VSRs have dual effects to maintain the balance between plant development and virus accumulation. Here, we summarize and discuss the multiple functions of the antiviral RNAi defense and VSRs, revealing the central hub regulators of VSRs in dynamically integrated connections between hosts and viruses.

PsHint1, associated with the G-protein α subunit PsGPA1, is required for the chemotaxis and pathogenicity of Phytophthora sojae.

Zoospore chemotaxis to soybean isoflavones is essential in the early stages of infection by the oomycete pathogen Phytophthora sojae. Previously, we have identified a G-protein α subunit encoded by PsGPA1 which regulates the chemotaxis and pathogenicity of P. sojae. In the present study, we used affinity purification to identify PsGPA1-interacting proteins, including PsHint1, a histidine triad (HIT) domain-containing protein orthologous to human HIT nucleotide-binding protein 1 (HINT1). PsHint1 interacted with both the guanosine triphosphate (GTP)- and guanosine diphosphate (GDP)-bound forms of PsGPA1. An analysis of the gene-silenced transformants revealed that PsHint1 was involved in the chemotropic response of zoospores to the isoflavone daidzein. During interaction with a susceptible soybean cultivar, PsHint1-silenced transformants displayed significantly reduced infectious hyphal extension and caused a strong cell death in plants. In addition, the transformants displayed defective cyst germination, forming abnormal germ tubes that were highly branched and exhibited apical swelling. These results suggest that PsHint1 not only regulates chemotaxis by interacting with PsGPA1, but also participates in a Gα-independent pathway involved in the pathogenicity of P. sojae.

The heat shock transcription factor PsHSF1 of Phytophthora sojae is required for oxidative stress tolerance and detoxifying the plant oxidative burst.

In the interaction between plant and microbial pathogens, reactive oxygen species (ROS) rapidly accumulate upon pathogen recognition at the infection site and play a central role in plant defence. However, the mechanisms that plant pathogens use to counteract ROS are still poorly understood especially in oomycetes, filamentous organisms that evolved independently from fungi. ROS detoxification depends on transcription factors (TFs) that are highly conserved in fungi but much less conserved in oomycetes. In this study, we identified the TF PsHSF1 that acts as a modulator of the oxidative stress response in the soybean stem and root rot pathogen Phytophthora sojae. We found that PsHSF1 is critical for pathogenicity in P. sojae by detoxifying the plant oxidative burst. ROS produced in plant defence can be detoxified by extracellular peroxidases and laccases which might be regulated by PsHSF1. Our study extends the understanding of ROS detoxification mechanism mediated by a heat shock TF in oomycetes.

Effect of Flumorph on F-Actin Dynamics in the Potato Late Blight Pathogen Phytophthora infestans.

Oomycetes are fungal-like pathogens that cause notorious diseases. Protecting crops against oomycetes requires regular spraying with chemicals, many with an unknown mode of action. In the 1990s, flumorph was identified as a novel crop protection agent. It was shown to inhibit the growth of oomycete pathogens including Phytophthora spp., presumably by targeting actin. We recently generated transgenic Phytophthora infestans strains that express Lifeact-enhanced green fluorescent protein (eGFP), which enabled us to monitor the actin cytoskeleton during hyphal growth. For analyzing effects of oomicides on the actin cytoskeleton in vivo, the P. infestans Lifeact-eGFP strain is an excellent tool. Here, we confirm that flumorph is an oomicide with growth inhibitory activity. Microscopic analyses showed that low flumorph concentrations provoked hyphal tip swellings accompanied by accumulation of actin plaques in the apex, a feature reminiscent of tips of nongrowing hyphae. At higher concentrations, swelling was more pronounced and accompanied by an increase in hyphal bursting events. However, in hyphae that remained intact, actin filaments were indistinguishable from those in nontreated, nongrowing hyphae. In contrast, in hyphae treated with the actin depolymerizing drug latrunculin B, no hyphal bursting was observed but the actin filaments were completely disrupted. This difference demonstrates that actin is not the primary target of flumorph.

Genome-wide analysis of pectate-induced gene expression in Botrytis cinerea: identification and functional analysis of putative d-galacturonate transporters.

The fungal plant pathogen Botrytis cinerea produces a spectrum of cell wall degrading enzymes for the decomposition of host cell wall polysaccharides and the consumption of the monosaccharides that are released. Especially pectin is an abundant cell wall component, and the decomposition of pectin by B. cinerea has been extensively studied. An effective concerted action of the appropriate pectin depolymerising enzymes, monosaccharide transporters and catabolic enzymes is important for complete d-galacturonic acid utilization by B. cinerea. In this study, we performed RNA sequencing to compare genome-wide transcriptional profiles between B. cinerea cultures grown in media containing pectate or glucose as sole carbon source. Transcript levels of 32 genes that are induced by pectate were further examined in cultures grown on six different monosaccharides, by means of quantitative RT-PCR, leading to the identification of 8 genes that are exclusively induced by d-galacturonic acid. Among these, the hexose transporter encoding genes Bchxt15 and Bchxt19 were functionally characterised. The subcellular location was studied of BcHXT15-GFP and BcHXT19-GFP fusion proteins expressed under control of their native promoter, in a B. cinerea wild-type strain. Both genes are expressed during growth on d-galacturonic acid and the fusion proteins are localized in plasma membranes and intracellular vesicles. Target gene knockout analysis revealed that BcHXT15 contributes to d-galacturonic acid uptake at pH 5∼5.6. The virulence of all B. cinerea hexose transporter mutants tested was unaltered on tomato and Nicotiana benthamiana leaves.