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Pathogen-associated molecular patterns (PAMPs) are conserved molecules that are crucial for normal life cycle of microorganisms. However, the diversity of microbial PAMPs is little known. During screening of cell-death-inducing factors from the necrotrophic fungus Valsa mali, we identified a novel PAMP VmE02 that is widely spread in oomycetes and fungi. Agrobacterium tumefaciens-mediated transient expression or infiltration of recombinant protein produced by Escherichia coli was performed to assay elicitor activity of the proteins tested. Virus-induced gene silencing in Nicotiana benthamiana was used to determine the components involved in VmE02-triggered cell death. The role of VmE02 in virulence and conidiation of V. mali were characterized by gene deletion and complementation. We found that VmE02, together with some of its homologues from both oomycete and fungal species, exhibited cell-death-inducing activity in N. benthamiana. VmE02-triggered cell death was shown to be dependent on BRI1-ASSOCIATED KINASE-1, SUPPRESSOR OF BIR1- 1, HSP90 and SGT1 in N. benthamiana. Deletion of VmE02 in V. mali greatly attenuated pathogen conidiation but not virulence, and treatment of N. benthamiana with VmE02 enhances plant resistance to Sclerotinia sclerotiorum and Phytophthora capsici. We conclude that VmE02 is a novel cross-kingdom PAMP produced by several fungi and oomycetes.

• Plant immunity consists of two arms: pathogen-associated molecular pattern (PAMP)- triggered immunity (PTI), induced by surface-localized receptors, and effector-triggered immunity (ETI), induced by intracellular receptors. Despite the little structural similarity, both receptor types activate similar responses with different dynamics. • To better understand phosphorylation events during ETI, we employed a phosphoproteomic screen using an inducible expression system of the bacterial effector avrRpt2 in Arabidopsis thaliana, and identified 109 differentially phosphorylated residues of membraneassociated proteins on activation of the intracellular RPS2 receptor. • Interestingly, several RPS2-regulated phosphosites overlap with sites that are regulated during PTI, suggesting that these phosphosites may be convergent points of both signaling arms. Moreover, some of these sites are residues of important defense components, including the NADPH oxidase RBOHD, ABC-transporter PEN3, calcium-ATPase ACA8, noncanonical Ga protein XLG2 and H⁺-ATPases. In particular, we found that S343 and S347 of RBOHD are common phosphorylation targets during PTI and ETI. Our mutational analyses showed that these sites are required for the production of reactive oxygen species during both PTI and ETI, and immunity against avirulent bacteria and a virulent necrotrophic fungus. • We provide, for the first time, large-scale phosphoproteomic data of ETI, thereby suggesting crucial roles of common phosphosites in plant immunity.

A microneedle array is an attractive option for a minimally invasive means to break through the skin barrier for efficient transdermal drug delivery. Here, we report the applications of solid polymer-based ion-conductive porous microneedles (PMN) containing interconnected micropores for improving iontophoresis, which is a technique of enhancing transdermal molecular transport by a direct current through the skin. The PMN modified with a charged hydrogel brings three innovative advantages in iontophoresis at once: (1) lowering the transdermal resistance by low-invasive puncture of the highly resistive stratum corneum, (2) transporting of larger molecules through the interconnected micropores, and (3) generating electroosmotic flow (EOF). In particular, the PMN-generated EOF greatly enhances the transdermal molecular penetration or extraction, similarly to the flow induced by external pressure. The enhanced efficiencies of the EOF-assisted delivery of a model drug (dextran) and of the extraction of glucose are demonstrated using a pig skin sample. Furthermore, the powering of the PMN-based transdermal EOF system by a built-in enzymatic biobattery (fructose / O 2 battery) is also demonstrated as a possible totally organic iontophoresis patch. Transdermal delivery has emerged as a preferred method of drug delivery. Here, the authors report on the application of porous polymer microneedles coupled with electroosmosis powered by enzymatic batteries for the transport of small and large molecules through the skin.

Pathogen-associated molecular patterns (PAMPs) are detected by plant pattern recognition receptors (PRRs), which gives rise to PAMP-triggered immunity (PTI). We characterized a novel fungal PAMP, Cell Death Inducing 1 (RcCDI1), identified in the Rhynchosporium commune transcriptome sampled at an early stage of barley (Hordeum vulgare) infection. The ability of RcCDI1 and its homologues from different fungal species to induce cell death in Nicotiana benthamiana was tested following agroinfiltration or infiltration of recombinant proteins produced by Pichia pastoris. Virus-induced gene silencing (VIGS) and transient expression of Phytophthora infestans effectors PiAVR3a and PexRD2 were used to assess the involvement of known components of PTI in N. benthamiana responses to RcCDI1. RcCDI1 was highly upregulated early during barley colonization with R. commune. RcCDI1 and its homologues from different fungal species, including Zymoseptoria tritici, Magnaporthe oryzae and Neurospora crassa, exhibited PAMP activity, inducing cell death in Solanaceae but not in other families of dicots or monocots. RcCDI1-triggered cell death was shown to require N. benthamiana Brassinosteroid insensitive 1-Associated Kinase 1 (NbBAK1), N. benthamiana suppressor of BIR1-1 (NbSOBIR1) and N. benthamiana SGT1 (NbSGT1), but was not suppressed by PiAVR3a or PexRD2. We report the identification of a novel Ascomycete PAMP, RcCDI1, recognized by Solanaceae but not by monocots, which activates cell death through a pathway that is distinct from that triggered by the oomycete PAMP INF1.

The phosphoinositide kinase PIP5K6 has recently been identified as a target for the mitogen-activated protein kinase (MAPK) MPK6. Phosphorylation of PIP5K6 inhibited the production of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂), impacting membrane trafficking and cell expansion in pollen tubes. Here, we analyzed whether MPK6 regulated PIP5K6 in vegetative Arabidopsis cells in response to the pathogen-associated molecular pattern (PAMP) flg22. Promoter-β-glucuronidase analyses and quantitative real-time reverse transcription polymerase chain reaction data show PIP5K6 expressed throughout Arabidopsis tissues. Upon flg22 treatment of transgenic protoplasts, the PIP5K6 protein was phosphorylated, and this modification was reduced for a PIP5K6 variant lacking MPK6-targeted residues, or in protoplasts from mpk6 mutants. Upon flg22 treatment of Arabidopsis plants, phosphoinositide levels mildly decreased and a fluorescent reporter for PtdIns(4,5)P₂ displayed reduced plasma membrane association, contrasting with phosphoinositide increases reported for abiotic stress responses. Flg22 treatment and chemical induction of the upstream MAPK kinase, MKK5, decreased phosphatidylinositol 4-phosphate 5-kinase activity in mesophyll protoplasts, indicating that the flg22-activated MAPK cascade limited PtdIns(4,5)P₂ production. PIP5K6 expression or PIP5K6 protein abundance changed only marginally upon flg22 treatment, consistent with post-translational control of PIP5K6 activity. PtdIns(4,5)P₂-dependent endocytosis of FM 4-64, PIN2 and the NADPH-oxidase RbohD were reduced upon flg22 treatment or MKK5 induction. Reduced RbohD-endocytosis was correlated with enhanced ROS production. We conclude that MPK6-mediated phosphorylation of PIP5K6 limits the production of a functional PtdIns(4,5)P₂ pool upon PAMP perception.

• Plant immune response is initiated upon the recognition of pathogen-associated molecular patterns such as elf18. Previously, we identified an Arabidopsis ELF18-INDUCED LONG NONCODING RNA 1 (ELENA1), as a positive transcriptional regulator of immune responsive genes. ELENA1 associated with Mediator subunit 19a (MED19a) to enhance enrichment of the complex on PATHOGENESIS-RELATED GENE 1 (PR1) promoter. • In vitro and in vivo RNA–protein interaction experiments showed that ELENA1 can also interact with FIBRILLARIN 2 (FIB2). Co-immunoprecipitation and bimolecular fluorescence complementation assay showed that FIB2 directly interacts with MED19a in nucleoplasm and nucleolus. Analysis of fib2 mutant showed that FIB2 functions as a negative transcriptional regulator for immune responsive genes, including PR1. • Genetic and biochemical analyses demonstrated that ELENA1 can dissociate the FIB2/MED19a complex and release FIB2 from PR1 promoter to enhance PR1 expression. • ELENA1 increases PR1 expression by evicting the repressor (FIB2) from the activator (MED19a). Our findings uncover an additional layer of complexity in the transcriptional regulation of plant immune responsive genes by long noncoding RNA.

Pattern-triggered immunity (PTI) is a plant defense response that relies on the perception of conserved microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs, respectively). Recently, it has been recognized that PTI restricts virus infection in plants; however, the nature of the viral or infection-induced PTI elicitors and the underlying signaling pathways are still unknown. As double-stranded RNAs (dsRNAs) are conserved molecular patterns associated with virus replication, we applied dsRNAs or synthetic dsRNA analogs to Arabidopsis thaliana and investigated PTI responses. We show that in vitro-generated dsRNAs, dsRNAs purified from virus-infected plants and the dsRNA analog polyinosinic–polycytidylic acid (poly(I:C)) induce typical PTI responses dependent on the co-receptor SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (SERK1), but independent of dicer-like (DCL) proteins in Arabidopsis. Moreover, dsRNA treatment of Arabidopsis induces SERK1-dependent antiviral resistance. Screening of Arabidopsis wild accessions demonstrates natural variability in dsRNA sensitivity. Our findings suggest that dsRNAs represent genuine PAMPs in plants, which induce a signaling cascade involving SERK1 and a specific dsRNA receptor. The dependence of dsRNAmediated PTI on SERK1, but not on DCLs, implies that dsRNA-mediated PTI involves membrane-associated processes and operates independently of RNA silencing. dsRNA sensitivity may represent a useful trait to increase antiviral resistance in cultivated plants.

Fusarium head blight is a destructive disease caused by Fusarium species. Little is known about the pathogenic molecular weapons of Fusarium graminearum. The gene encoding a small secreted protein, Fg02685, in F. graminearum was found to be upregulated during wheat head infection. Knockout mutation of Fg02685 reduced the growth and development of Fusarium in wheat spikes. Transient expression of Fg02685 or recombinant protein led to plant cell death in a BAK1‐ and SOBIR1‐independent system. Fg02685 was found to trigger plant basal immunity by increasing the deposition of callose, the accumulation of reactive oxygen species (ROS), and the expression of defence‐related genes. The Fg02685 signal peptide was required for the plant's apoplast accumulation and induces cell death, indicating Fg02685 is a novel conserved pathogen‐associated molecular pattern. Moreover, its homologues are widely distributed in oomycetes and fungal pathogens and induced cell death in tobacco. The conserved α‐helical motif at the N‐terminus was necessary for the induction of cell death. Moreover, a 32‐amino‐acid peptide, Fg02685 N‐terminus peptide 32 (FgNP32), was essential for the induction of oxidative burst, callose deposition, and mitogen‐activated protein kinase signal activation in plants. Prolonged exposure to FgNP32 enhanced the plant's resistance to Fusarium and Phytophthora. This study provides new approaches for an environment‐friendly control strategy for crop diseases by applying plant immune inducers to strengthen broad‐spectrum disease resistance in crops. A 32‐amino‐acid peptide (FgNP32) of a Fusarium graminearum‐secreted protein is sufficient to activate plant immunity and enhance the plant's resistance to Fusarium and Phytophthora.

Evidence is emerging that plant‐parasitic nematodes can secrete effectors to interfere with the host immune response, but it remains unknown how these effectors can conquer host immune responses. Here, we depict a novel effector, MjTTL5, that could suppress plant immune response. Immunolocalization and transcriptional analyses showed that MjTTL5 is expressed specifically within the subventral gland of Meloidogyne javanica and up‐regulated in the early parasitic stage of the nematode. Transgenic Arabidopsis lines expressing MjTTL5 were significantly more susceptible to M. javanica infection than wild‐type plants, and vice versa, in planta silencing of MjTTL5 substantially increased plant resistance to M. javanica. Yeast two‐hybrid, coimmunoprecipitation and bimolecular fluorescent complementation assays showed that MjTTL5 interacts specifically with Arabidopsis ferredoxin : thioredoxin reductase catalytic subunit (AtFTRc), a key component of host antioxidant system. The expression of AtFTRc is induced by the infection of M. javanica. Interaction between AtFTRc and MjTTL could drastically increase host reactive oxygen species‐scavenging activity, and result in suppression of plant basal defenses and attenuation of host resistance to the nematode infection. Our results demonstrate that the host ferredoxin : thioredoxin system can be exploited cunningly by M. javanica, revealing a novel mechanism utilized by plant–parasitic nematodes to subjugate plant innate immunity and thereby promoting parasitism.

Secreted small cysteine‐rich proteins (SCPs) play a critical role in modulating host immunity in plant–pathogen interactions. Bioinformatic analyses showed that the fungal pathogen Verticillium dahliae encodes more than 100 VdSCPs, but their roles in host–pathogen interactions have not been fully characterized. Transient expression of 123 VdSCP‐encoding genes in Nicotiana benthamiana identified three candidate genes involved in host–pathogen interactions. The expression of these three proteins, VdSCP27, VdSCP113, and VdSCP126, in N. benthamiana resulted in cell death accompanied by a reactive oxygen species burst, callose deposition, and induction of defence genes. The three VdSCPs mainly localized to the periphery of the cell. BAK1 and SOBIR1 (associated with receptor‐like protein) were required for the immunity triggered by these three VdSCPs in N. benthamiana. Site‐directed mutagenesis showed that cysteine residues that form disulphide bonds are essential for the functioning of VdSCP126, but not VdSCP27 and VdSCP113. VdSCP27, VdSCP113, and VdSCP126 individually are not essential for V. dahliae infection of N. benthamiana and Gossypium hirsutum, although there was a significant reduction of virulence on N. benthamiana and G. hirsutum when inoculated with the VdSCP27/VdSCP126 double deletion strain. These results illustrate that the SCPs play a critical role in the V. dahliae–plant interaction via an intrinsic virulence function and suppress immunity following infection. Small cysteine‐rich proteins secreted by Verticillium dahliae play critical roles in interactions with hosts via an intrinsic virulence function and can suppress immunity following infection.