Despite the recent discovery that trehalose synthesis is important for plant development and abiotic stress tolerance, the effects of trehalose on biotic stress responses remain relatively unknown. In this study, we demonstrate that TREHALOSE PHOSPHATE SYNTHASE 5 (TPS5)-dependent trehalose metabolism regulates Arabidopsis thaliana defenses against pathogens (necrotrophic Botrytis cinerea and biotrophic Pseudomonas syringae). Pathogen infection increased trehalose levels and upregulated TPS5 expression. Application of exogenous trehalose significantly improved plant defenses against B. cinerea, but increased the susceptibility of plants to P. syringae. We demonstrate that elevated trehalose biosynthesis, in transgenic plants over-expressing TPS5, also increased the susceptibility to P. syringae, but decreased the disease symptoms caused by B. cinerea. The knockout of TPS5 prevented the accumulation of trehalose and enhanced defense responses against P. syringae. Additionally, we observed that a TPS5-interacting protein (multiprotein bridging factor 1c) was required for induced expression of TPS5 during pathogen infections. Furthermore, we show that trehalose promotes P. syringae growth and disease development, via a mechanism involving suppression of the plant defense gene, Pathogenesis-Related Protein 1. These findings provide insight into the function of TPS5-dependent trehalose metabolism in plant basal defense responses.

MicroRNAs (miRNAs) play important roles in rice response to Magnaporthe oryzae, the causative agent of rice blast disease. Studying the roles of rice miRNAs is of great significance for the disease control. Osa‐miR167d belongs to a conserved miRNA family targeting auxin responsive factor (ARF) genes that act in developmental and stress‐induced responses. Here, we show that Osa‐miR167d plays a negative role in rice immunity against M. oryzae by suppressing its target gene. The expression of Osa‐miR167d was significantly suppressed in a resistant accession at and after 24 h post inoculation (hpi), however, its expression was significantly increased at 24 hpi in the susceptible accession upon M. oryzae infection. Transgenic rice lines over‐expressing Osa‐miR167d were highly susceptible to multiple blast fungal strains. By contrast, transgenic lines expressing a target mimicry to block Osa‐miR167d enhanced resistance to rice blast disease. In addition, knocking out the target gene ARF12 led to hyper‐susceptibility to multiple blast fungal strains. Taken together, our results indicate that Osa‐miR167d negatively regulate rice immunity to facilitate the infection of M. oryzae by downregulating ARF12. Thus, Osa‐miR167d‐ARF12 regulatory module could be valuable in improvement of blast‐disease resistance.

Isochorismate synthase (ICS) plays an essential role in the accumulation of salicylic acid (SA) and plant disease resistance. Diseases caused by Botryosphaeria dothidea affect apple yields. Thus, it is important to understand the role of ICS1 in disease resistance to B. dothidea in apple. In this study, SA treatment enhanced the resistance to B. dothidea. MdICS1 was induced by B. dothidea and enhanced the resistance to B. dothidea. MdICS1 promoter analysis indicated that the W‐box was vital for the response to B. dothidea treatment. MdWRKY15 was found to interact with the W‐box using yeast one‐hybrid screening. Subsequently, the interaction was confirmed by EMSA, yeast one‐hybrid, ChIP‐PCR, and quantitative PCR assays. Moreover, luciferase and GUS analysis further indicated that MdICS1 was transcriptionally activated by MdWRKY15. Finally, we found the function of MdWRKY15 in the resistance to B. dothidea was partially dependent on MdICS1 from the phenotype of transgenic apples and calli. In summary, we revealed that MdWRKY15 activated the transcription of MdICS1 by directly binding to its promoter to increase the accumulation of SA and the expression of disease‐related genes, thereby resulting in the enhanced resistance to B. dothidea in the SA biosynthesis pathway.

Trichoderma biocontrol strains establish a complex network of interactions with plants, in which diverse fungal molecules are involved in the recognition of these fungi as nonpathogenic organisms. These molecules act as microbial‐associated molecular patterns that trigger plant responses. Previous studies have reported the importance of ergosterol produced by Trichoderma spp. for the ability of these fungi to induce plant growth and defenses. In addition, squalene, a sterol biosynthetic intermediate, seems to play an important role in these interactions. Here, we analyzed the effect of different concentrations of ergosterol and squalene on tomato (Solanum lycopersicum ) growth and on the transcription level of defense‐ and growth‐related genes. We used an RNA‐seq strategy to identify several tomato genes encoding predicted pattern recognition receptor proteins or WRKY transcription factors, both of which are putatively involved in the perception and response to ergosterol and squalene. Finally, an analysis of Arabidopsis thaliana mutants lacking the genes homologous to these tomato candidates led to the identification of a WRKY40 transcription factor that negatively regulates salicylic acid‐related genes and positively regulates ethylene‐ and jasmonate‐related genes in the presence of ergosterol and squalene.

Plants have evolved multiple defense strategies to cope with pathogens, among which plant immune signaling that relies on cell‐surface localized and intracellular receptors takes fundamental roles. Exciting breakthroughs were made recently on the signaling mechanisms of pattern recognition receptors (PRRs) and intracellular nucleotide‐binding site (NBS) and leucine‐rich repeat (LRR) domain receptors (NLRs). This review summarizes the current view of PRRs activation, emphasizing the most recent discoveries about PRRs’ dynamic regulation and signaling mechanisms directly leading to downstream molecular events including mitogen‐activated protein kinase (MAPK) activation and calcium (Ca²⁺) burst. Plants also have evolved intracellular NLRs to perceive the presence of specific pathogen effectors and trigger more robust immune responses. We also discuss the current understanding of the mechanisms of NLR activation, which has been greatly advanced by recent breakthroughs including structures of the first full‐length plant NLR complex, findings of NLR sensor‐helper pairs and novel biochemical activity of Toll/interleukin‐1 receptor (TIR) domain.

Secondary plant metabolites, represented by indole glucosinolates (IGS) and camalexin, play important roles in Arabidopsis immunity. Previously, we demonstrated the importance of MPK3 and MPK6, two closely related MAPKs, in regulating Botrytis cinerea (Bc)‐induced IGS and camalexin biosynthesis. Here we report that CPK5 and CPK6, two redundant calcium‐dependent protein kinases (CPKs), are also involved in regulating the biosynthesis of these secondary metabolites. The loss‐of‐function of both CPK5 and CPK6 compromises plant resistance to Bc. Expression profiling of CPK5‐VK transgenic plants, in which a truncated constitutively active CPK5 is driven by a steroid‐inducible promoter, revealed that biosynthetic genes of both IGS and camalexin pathways are coordinately upregulated after the induction of CPK5‐VK, leading to high‐level accumulation of camalexin and 4‐methoxyindole‐3‐yl‐methylglucosinolate (4MI3G). Induction of camalexin and 4MI3G, as well as the genes in their biosynthesis pathways, is greatly compromised in cpk5 cpk6 mutant in response to Bc. In a conditional cpk5 cpk6 mpk3 mpk6 quadruple mutant, Bc resistance and induction of IGS and camalexin are further reduced in comparison to either cpk5 cpk6 or conditional mpk3 mpk6 double mutant, suggesting that both CPK5/CPK6 and MPK3/MPK6 signaling pathways contribute to promote the biosynthesis of 4MI3G and camalexin in defense against Bc.

Disruption of the MEKK1‐MKK1/MKK2‐MPK4 kinase cascade leads to activation of immunity mediated by the nucleotide‐binding leucine‐rich repeat (NLR) immune receptor SUMM2, which monitors the phosphorylation status of CRCK3. Here we report that two receptor‐like kinases (RLKs), MDS1, and MDS2, function redundantly to promote SUMM2‐mediated immunity. Activation of SUMM2‐mediated immunity is dependent on MDS1, and to a less extent on MDS2. MDS1 associates with CRCK3 in planta and can phosphorylate CRCK3 in vitro, suggesting that it may target CRCK3 to positively regulate SUMM2‐mediated signaling. Our finding highlights a new defense mechanism where RLKs promote NLR‐mediated immunity.

Filamentous fungal pathogens secrete effectors that modulate host immunity and facilitate infection. Fusarium graminearum is an important plant pathogen responsible for various devastating diseases. However, little is known about the function of effector proteins secreted by F. graminearum. Herein, we identified several effector candidates in the F. graminearum secretome. Among them, the secreted ribonuclease Fg12 was highly upregulated during the early stages of F. graminearum infection in soybean; its deletion compromised the virulence of F. graminearum. Transient expression of Fg12 in Nicotiana benthamiana induced cell death in a light‐dependent manner. Fg12 possessed ribonuclease (RNase) activity, degrading total RNA. The enzymatic activity of Fg12 was required for its cell death‐promoting effects. Importantly, the ability of Fg12 to induce cell death was independent of BAK1/SOBIR1, and treatment of soybean with recombinant Fg12 protein induced resistance to various pathogens, including F. graminearum and Phytophthora sojae. Overall, our results provide evidence that RNase effectors not only contribute to pathogen virulence but also induce plant cell death.

Mitogen‐activated protein kinase (MAPK) cascades are highly conserved signaling modules that regulate plant immune responses. The Arabidopsis thaliana Raf‐like MAPK kinase kinase ENHANCED DISEASE RESISTANCE1 (EDR1) is a key negative regulator of plant immunity that affects the protein levels of MKK4 and MKK5, two important MAPK cascade members, but the underlying mechanism is poorly understood. Here, genome‐wide phosphorylation analysis demonstrated that the E3 ligase KEEP ON GOING (KEG) is phosphorylated in the edr1 mutant but not the wild type, suggesting that EDR1 negatively affects KEG phosphorylation. The identified phosphorylation sites in KEG appear to be important for its accumulation. The keg‐4 mutant, a previously identified edr1 suppressor, enhances susceptibility to the powdery mildew pathogen Golovinomyces cichoracearum. In addition, MKK4 and MKK5 protein levels are reduced in the keg‐4 mutant. Furthermore, we demonstrate that MKK4 and MKK5 associate with full‐length KEG, but not with truncated KEG‐RK or KEG‐RKA, and that KEG ubiquitinates and mediates the degradation of MKK4 and MKK5. Taken together, these results indicate that MKK4 and MKK5 protein levels are regulated by KEG via ubiquitination, uncovering a mechanism by which plants fine‐tune immune responses by regulating the homeostasis of key MAPK cascade members via ubiquitination and degradation.

Study on the regulation of broad‐spectrum resistance is an active area in plant biology. RESISTANCE TO POWDERY MILDEW 8.1 (RPW8.1) is one of a few broad‐spectrum resistance genes triggering the hypersensitive response (HR) to restrict multiple pathogenic infections. To address the question how RPW8.1 signaling is regulated, we performed a genetic screen and tried to identify mutations enhancing RPW8.1‐mediated HR. Here, we provided evidence to connect an annexin protein with RPW8.1‐mediated resistance in Arabidopsis against powdery mildew. We isolated and characterized Arabidopsis b7‐6 mutant. A point mutation in b7‐6 at the At5g12380 locus resulted in an amino acid substitution in ANNEXIN 8 (AtANN8). Loss‐of‐function or RNA‐silencing of AtANN8 led to enhanced expression of RPW8.1, RPW8.1‐dependent necrotic lesions in leaves, and defense against powdery mildew. Conversely, over‐expression of AtANN8 compromised RPW8.1‐mediated disease resistance and cell death. Interestingly, the mutation in AtANN8 enhanced RPW8.1‐triggered H₂O₂. In addition, mutation in AtANN8 led to hypersensitivity to salt stress. Together, our data indicate that AtANN8 is involved in multiple stress signaling pathways and negatively regulates RPW8.1‐mediated resistance against powdery mildew and cell death, thus linking ANNEXIN's function with plant immunity.

The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three‐dimensional (3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus (CMV) or tobacco necrosis virus A Chinese isolate (TNV‐A^C), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck‐like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1a and 2a and TNV‐A^C auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast. Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant (+) RNA virus replication complexes.

The development of durable and broad‐spectrum resistance is an economical and eco‐friendly approach to control crop diseases for sustainable agricultural production. Emerging knowledge of the molecular basis of pathogenesis and plant–pathogen interactions has contributed to the development of novel pathogen‐informed breeding strategies beyond the limits imposed by conventional breeding. Here, we review the current status of pathogen‐assisted resistance‐related gene cloning. We also describe how pathogen effector proteins can be used to identify resistance resources and to inform cultivar deployment. Finally, we summarize the main approaches for pathogen‐directed plant improvement, including transgenesis and genome editing. Thus, we describe the emerging role of pathogen‐related studies in the breeding of disease‐resistant varieties, and propose innovative pathogen‐informed strategies for future applications.

Plant nucleotide‐binding, leucine‐rich repeat receptors (NLRs) perceive pathogen avirulence effectors and activate defense responses. Nucleotide‐binding, leucine‐rich repeat receptors are classified into coiled‐coil (CC)‐containing and Toll/interleukin‐1 receptor (TIR)‐containing NLRs. Recent advances suggest that NLR CC domains often function in signaling activation, especially for induction of cell death. In this review, we outline our current understanding of NLR CC domains, including their diversity/classification and structure, their roles in cell death induction, disease resistance, and interaction with other proteins. Furthermore, we provide possible directions for future work.

DNA methylation is an epigenetic mark important for genome stability and gene expression. In Arabidopsis thaliana, the 5‐methylcytosine DNA glycosylase/demethylase DEMETER (DME) controls active DNA demethylation during the reproductive stage; however, the lethality of loss‐of‐function dme mutations has made it difficult to assess DME function in vegetative tissues. Here, we edited DME using clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR‐associated protein 9 and created three weak dme mutants that produced a few viable seeds. We also performed central cell‐specific complementation in a strong dme mutant and combined this line with mutations in the other three Arabidopsis demethylase genes to generate the dme ros1 dml2 dml3 (drdd) quadruple mutant. A DNA methylome analysis showed that DME is required for DNA demethylation at hundreds of genomic regions in vegetative tissues. A transcriptome analysis of the drdd mutant revealed that DME and the other three demethylases are important for plant responses to biotic and abiotic stresses in vegetative tissues. Despite the limited role of DME in regulating DNA methylation in vegetative tissues, the dme mutants showed increased susceptibility to bacterial and fungal pathogens. Our study highlights the important functions of DME in vegetative tissues and provides valuable genetic tools for future investigations of DNA demethylation in plants.

Class III peroxidases (CIII Prxs) play critical roles in plant immunity by scavenging reactive oxygen species (ROS). However, the functions of CIII Prxs in rice (Oryza sativa L.) immunity are largely unexplored. Here, we report a Prx precursor, OsPrx30, that is responsive to the bacterial blight Xanthomonas oryzae pv. oryzae (Xoo). OsPrx30 was primarily expressed in rice roots, leaves, and stems, and its protein product was mainly localized at the endoplasmic reticulum. Overexpression of OsPrx30 enhanced the plant's susceptibility to Xoo by maintaining a high level of peroxidase (POD) activity and reducing the content of H₂O₂, whereas depletion of OsPrx30 had the opposite effects. Furthermore, we identified an AT‐hook transcription factor, OsATH1, that is specifically bound to the OsPrx30 promoter. As observed in plants overexpressing OsPrx30, depletion of OsATH1 enhanced susceptibility to Xoo. Finally, we demonstrated that depletion of OsATH1 increased histone H3 acetylation at the AT‐rich region of the OsPrx30 promoter. Taken together, these results reveal a mechanism underlying the POD‐induced natural resistance to bacterial diseases and suggest a model for transcription regulation of Prx genes in rice.

As sessile organisms, plants are exposed to pathogen invasions and environmental fluctuations. To overcome the challenges of their surroundings, plants acquire the potential to sense endogenous and exogenous cues, resulting in their adaptability. Hence, plants have evolved a large collection of plasma membrane‐resident receptors, including RECEPTOR‐LIKE KINASEs (RLKs) and RECEPTOR‐LIKE PROTEINs (RLPs) to perceive those signals and regulate plant growth, development, and immunity. The ability of RLKs and RLPs to recognize distinct ligands relies on diverse categories of extracellular domains evolved. Co‐regulatory receptors are often required to associate with RLKs and RLPs to facilitate cellular signal transduction. RECEPTOR‐LIKE CYTOPLASMIC KINASEs (RLCKs) also associate with the complex, bifurcating the signal to key signaling hubs, such as MITOGEN‐ACTIVATED PROTEIN KINASE (MAPK) cascades, to regulate diverse biological processes. Here, we discuss recent knowledge advances in understanding the roles of RLKs and RLPs in plant growth, development, and immunity, and their connection with co‐regulatory receptors, leading to activation of diverse intracellular signaling pathways.

Plants are colonized by various microorganisms in natural environments. While many studies have demonstrated key roles of the rhizosphere microbiota in regulating biological processes such as nutrient acquisition and resistance against abiotic and biotic challenges, less is known about the role of the phyllosphere microbiota and how it is established and maintained. This review provides an update on current understanding of phyllosphere community assembly and the mechanisms by which plants and microbes establish the phyllosphere microbiota for plant health.

Lysine 2‐hydroxyisobutyrylation (K_hib) is a newly identified post‐translational modification (PTM) that plays important roles in transcription and cell proliferation in eukaryotes. However, its function remains unknown in phytopathogenic fungi. Here, we performed a comprehensive assessment of K_hib in the rice false smut fungus Ustilaginoidea virens, using Tandem Mass Tag (TMT)‐based quantitative proteomics approach. A total of 3 426 K_hib sites were identified in 977 proteins, suggesting that K_hib is a common and complex PTM in U. virens. Our data demonstrated that the 2‐hydroxyisobutyrylated proteins are involved in diverse biological processes. Network analysis of the modified proteins revealed a highly interconnected protein network that included many well‐studied virulence factors. We confirmed that the Zn‐binding reduced potassium dependency3‐type histone deacetylase (UvRpd3) is a major enzyme that removes 2‐hydroxyisobutyrylation and acetylation in U. virens. Notably, mutations of K_hib sites in the mitogen‐activated protein kinase (MAPK) UvSlt2 significantly reduced fungal virulence and decreased the enzymatic activity of UvSlt2. Molecular dynamics simulations demonstrated that 2‐hydroxyisobutyrylation in UvSlt2 increased the hydrophobic solvent‐accessible surface area and thereby affected binding between the UvSlt2 enzyme and its substrates. Our findings thus establish K_hib as a major post‐translational modification in U. virens and point to an important role for K_hib in the virulence of this phytopathogenic fungus.

The post-translational modification lysine 2-hydroxyisobutyrylation (K_hib) plays an important role in gene transcription, metabolism, and enzymatic activity. K_hib sites have been identified in rice (Oryza sativa). However, the K_hib status of proteins in rice flowers during pathogen infection remains unclear. Here, we report a comprehensive identification of K_hib-modified proteins in rice flowers, and the changes in these proteins during infection with the fungal pathogen Ustilaginoidea virens. By using a tandem mass tag-based quantitative proteomics approach, we identified 2,891 K_hib sites on 964 proteins in rice flowers. Our data demonstrated that 2-hydroxyisobutyrylated proteins are involved in diverse biological processes. K_hib levels were substantially reduced upon infection with U. virens. Chromatin immunoprecipitation polymerase chain reaction (PCR) and reverse transcription quantitative PCR analyses revealed that histone K_hib is involved in the expression of disease-resistance genes. More importantly, most quantified sites on core histones H3 were downregulated upon U. virens infection. In addition, the histone deacetylases HDA705, HDA716, SRT1, and SRT2 are involved in the removal of K_hib marks in rice. HDA705 was further confirmed to negatively regulate rice disease resistance to pathogens U. virens, Magnaporthe oryzae, and Xanthomonas oryzae pv. oryzae (Xoo). Our data suggest that U. virens could modulate K_hib in rice flowers during infection.

The rhizosheath, a layer of soil grains that adheres firmly to roots, is beneficial for plant growth and adaptation to drought environments. Switchgrass is a perennial C4 grass which can form contact rhizosheath under drought conditions. In this study, we characterized the microbiomes of four different rhizocompartments of two switchgrass ecotypes (Alamo and Kanlow) grown under drought or well-watered conditions via 16S ribosomal RNA amplicon sequencing. These four rhizocompartments, the bulk soil, rhizosheath soil, rhizoplane, and root endosphere, harbored both distinct and overlapping microbial communities. The root compartments (rhizoplane and root endosphere) displayed low-complexity communities dominated by Proteobacteria and Firmicutes. Compared to bulk soil, Cyanobacteria and Bacteroidetes were selectively enriched, while Proteobacteria and Firmicutes were selectively depleted, in rhizosheath soil. Taxa from Proteobacteria or Firmicutes were specifically selected in Alamo or Kanlow rhizosheath soil. Following drought stress, Citrobacter and Acinetobacter were further enriched in rhizosheath soil, suggesting that rhizosheath microbiome assembly is driven by drought stress. Additionally, the ecotype-specific recruitment of rhizosheath microbiome reveals their differences in drought stress responses. Collectively, these results shed light on rhizosheath microbiome recruitment in switchgrass and lay the foundation for the improvement of drought tolerance in switchgrass by regulating the rhizosheath microbiome.

Interactions and co-evolution between plants and herbivorous insects are critically important in agriculture. Brown planthopper (BPH) is the most severe insect of rice, and the biotypes adapt to feed on different rice genotypes. Here, we present genomics analyses on 1,520 global rice germplasms for resistance to three BPH biotypes. Genome-wide association studies identified 3,502 single nucleotide polymorphisms (SNPs) and 59 loci associated with BPH resistance in rice. We cloned a previously unidentified gene Bph37 that confers resistance to BPH. The associated loci showed high nucleotide diversity. Genome-wide scans for trans-species polymorphisms revealed ancient balancing selection at the loci. The secondarily evolved insect biotypes II and III exhibited significantly higher virulence and overcame more rice varieties than the primary biotype I. In response, more SNPs and loci evolved in rice for resistance to biotypes II and III. Notably, three exceptional large regions with high SNP density and resistance-associated loci on chromosomes 4 and 6 appear distinct between the resistant and susceptible rice varieties. Surprisingly, these regions in resistant rice might have been retained from wild species Oryza nivara. Our findings expand the understanding of long-term interactions between rice and BPH and provide resistance genes and germplasm resources for breeding durable BPH-resistant rice varieties.