Bioactive secondary metabolites from fungi, including Trichoderma, are an excellent source of plant biostimulants. Although production of novel biostimulants from known microbes is critical, challenging them may produce novel bioactive compounds. With this hypothesis, the study used live Fusarium chlamydosporum (FOL7) culture as the inducer during T. harzianum (IF63) growth in broth. Plate assays and gas chromatography-mass spectrometry (GC-MS) analysis were used to characterise the metabolites. Microscopy, pot experiments and, biochemical estimations of the defence-related enzymes in tomato plants established the biostimulant activity of the induced Trichoderma metabolites. Fungal crude metabolites (FCM) obtained from IF63+FOL7 extracts (TF.ex) showed increased antimicrobial activity. TF.ex at 50 μg mL-1, inhibited the FOL7 growth by 68.33% compared to the Trichoderma alone extract. Scanning electron microscopy (SEM) revealed morphological disruption of FOL7 mycelia by TF.ex. GC-MS analysis of the extracts revealed the presence of approximately 64 compounds, of which at least 13 were detected explicitly in TF.ex. Methyl (3-oxo-2-pentylcyclopentyl) acetate (Methyl dihydrojasmonate), a lipid functionally related to jasmonic acid, was the major metabolite (∼21%) present in TF.ex. Tomato seed dressing with TF.ex promoted plant growth and induced systemic resistance against FOL7 compared to alone Trichoderma and Fusarium extracts. The TF.ex treatment increased the superoxide dismutase (33%) and catalase activity by 2.5-fold in tomato plants. The study concludes that fungal secondary metabolites may be modulated by providing appropriate challenges to produce effective metabolite-based biostimulants for agricultural applications.

Plants possess cell surface-localized immune receptors that detect microbe-associated molecular patterns (MAMPs) and initiate defenses that provide effective resistance against microbial pathogens. Many MAMP-induced signaling pathways and cellular responses are known, yet how pattern-triggered immunity (PTI) limits pathogen growth in plants is poorly understood. Through a combined metabolomics and genetics approach, we discovered that plant-exuded proline is a virulence-inducing signal and nutrient for the bacterial pathogen Pseudomonas syringae, and that MAMP-induced depletion of proline from the extracellular spaces of Arabidopsis leaves directly contributes to PTI against P. syringae. We further show that MAMP-induced depletion of extracellular proline requires the amino acid transporter Lysine Histidine Transporter 1 (LHT1). This study demonstrates that depletion of a single extracellular metabolite is an effective component of plant induced immunity. Given the important role for amino acids as nutrients for microbial growth, their depletion at sites of infection may be a broadly effective means for defense against many pathogens.

Bacterial extracellular vesicles (BEVs) are released from the surface of bacterial cells and contain a diverse molecular cargo. Studies conducted primarily with bacterial pathogens of mammals have shown that BEVs are involved in multiple processes such as cell-cell communication, the delivery of RNA, DNA, and proteins to target cells, protection from stresses, manipulation of host immunity, and other functions. Until a decade ago, the roles of BEVs in plant-bacteria interactions were barely investigated. However, recent studies have shown that BEVs of plant pathogens possess similar functions as their mammalian pathogen counterparts, and more research is now devoted to study their roles and interactions with plants. In the following methods chapter, we provide five well-validated assays to examine the interaction of BEVs with the plant immune system. These assays rely on different markers or immune outputs, which indicate the activation of plant immunity (defense marker gene expression, reactive oxygen species burst, seedling inhibition). Furthermore, we offer assays that directly evaluate the priming of the immune system following BEV challenge and the effectiveness of its response to subsequent local or systemic infection. Altogether, these assays provide a thorough examination to the interactions of BEVs and the plant immune system.

An integral part of plant immunity is transcription reprogramming by concerted action of specific transcription factors that activate or repress genes through recruitment or release of RNA polymerase II (Pol II). Pol II is assembled into Pol II holoenzyme at the promoters through association with a group of general transcription factors including transcription factor IIB (TFIIB) to activate transcription. Unlike other eukaryotic organisms, plants have a large family of TFIIB-related proteins with 15 members in Arabidopsis including several plant-specific TFIIB-related proteins (BRPs). Molecular genetic analysis has revealed important roles of some BRPs in plant reproductive processes. In this study, we report that Arabidopsis knockout mutants for BRP1, the founding member of the BRP protein family, were normal in growth and development, but were hypersusceptible to the bacterial pathogen Psuedomonas syringae. The enhanced susceptibility of the brp1 mutants was associated with reduced expression of salicylic acid (SA) biosynthetic gene ISOCHORISMATE SYNTHASE 1 (ICS1) and SA-responsive PATHOGENESIS-RELATED (PR) genes. Pathogen-induced SA accumulation was reduced in the brp1 mutants and exogenous SA rescued the brp1 mutants for resistance to the bacterial pathogen. In uninfected plants, BRP1 was primarily associated with the plastids but pathogen infection induced its accumulation in the nucleus. BRP1 acted as a transcription activator in plant cells and binded to the promoter of ICS1. These results collectively indicate that BRP1 is a functionally specialized transcription factor that increasingly accumulates in the nucleus in response to pathogen infection to promote defense gene expression.

Chloroplasts are key players in photosynthesis and immunity against microbial pathogens. However, the precise and timely regulatory mechanisms governing the control of photosynthesis-associated nuclear genes (PhANGs) expression in plant immunity remain largely unknown. Here we report that TaPIR1, a Pst-induced RING-finger E3 ubiquitin ligase, negatively regulates Pst resistance by specifically interacting with TaHRP1, an atypical transcription factor histidine-rich protein. TaPIR1 ubiquitinates the lysine residues K131 and K136 in TaHRP1 to regulate its stability. TaHRP1 directly binds to the TaHRP1-binding site elements within the PhANGs promoter to activate their transcription via the histidine-rich domain of TaHRP1. PhANGs expression induces the production of chloroplast-derived ROS. Although knocking out TaHRP1 reduces Pst resistance, TaHRP1 overexpression contributes to photosynthesis, and chloroplast-derived ROS production, and improves disease resistance. TaPIR1 expression inhibits the downstream activation of TaHRP1 and TaHRP1-induced ROS accumulation in chloroplasts. Overall, we show that the TaPIR1-mediated ubiquitination and degradation of TaHRP1 alters PhANGs expression to disrupt chloroplast function, thereby increasing plant susceptibility to Pst.

The indica rice variety Huizhan shows elite traits of disease resistance and heat tolerance. However, the underlying genetic basis of these traits is not fully understood due to limited genomic resources. Here, we used Nanopore long-read and next-generation sequencing technologies to generate a chromosome-scale genome assembly of Huizhan. Comparative genomics analysis uncovered a large chromosomal inversion and expanded gene families that are associated with plant growth, development and stress responses. Functional rice blast resistance genes, including Pi2, Pib and Ptr, and bacterial blight resistance gene Xa27, contribute to disease resistance of Huizhan. Furthermore, integrated genomics and transcriptomics analyses showed that OsHIRP1, OsbZIP60, the SOD gene family, and various transcription factors are involved in heat tolerance of Huizhan. The high-quality genome assembly and comparative genomics results presented in this study facilitate the use of Huizhan as an elite parental line in developing rice varieties adapted to disease pressure and climate challenges.

Isochorismate-derived metabolism enables biosynthesis of the plant defence hormone salicylic acid (SA) and its derivatives. In Arabidopsis thaliana, the stress-induced accumulation of SA depends on ISOCHORISMATE SYNTHASE1 (ICS1), and also requires the presumed isochorismate transporter ENHANCED DISEASE SUSCEPTIBILITY5 (EDS5) and the GH3 enzyme avrPphB SUSCEPTIBLE3 (PBS3). By comparative metabolite and structural analyses, we identified several hitherto unreported ICS1- and EDS5-dependent, biotic stress-inducible Arabidopsis metabolites. These involve meta-substituted SA derivatives (5-formyl-SA, 5-carboxy-SA, 5-carboxymethyl-SA), their benzoic acid (BA) analogues (3-formyl-BA, 3-carboxy-BA, 3-carboxymethyl-BA) and, besides the previously detected salicyloyl-aspartate (SA-Asp), the ester conjugate salicyloyl-malate (SA-Mal). SA functions as a biosynthetic precursor for SA-Mal and SA-Asp, but not for the meta-substituted SA- and BA-derivatives, which accumulate to moderate levels at later stages of bacterial infection. Interestingly, Arabidopsis leaves possess oxidising activity to effectively convert meta-formyl- into meta-carboxy-SA/BAs. In contrast to SA, exogenously applied meta-substituted SA/BA-derivatives and SA-Mal exert moderate impact on plant immunity and defence-related gene expression. While the isochorismate-derived metabolites are negatively regulated by the SA receptor NON-EXPRESSOR OF PR GENES1, SA conjugates (SA-Mal, SA-Asp, SA-glucose conjugates) and meta-substituted SA/BA-derivatives are oppositely affected by PBS3. Notably, our data indicate a PBS3-independent path to isochorismate-derived SA at later stages of bacterial infection, which does not considerably impact immune-related characteristics. Moreover, our results argue against a previously proposed role of EDS5 in the biosynthesis of the immune signal N-hydroxypipecolic acid and associated transport processes. We propose a significantly extended biochemical scheme of plant isochorismate metabolism that involves an alternative generation mode for benzoate- and salicylate-derivatives.

Stomatal movement plays a critical role in plant immunity by limiting the entry of pathogens. OPEN STOMATA 1 (OST1) is a key component that mediates stomatal closure in plants, however, how OST1 functions in response to pathogens is not well understood. RECEPTOR-LIKE KINASE 902 (RLK902) phosphorylates BRASSINOSTEROID-SIGNALING KINASE 1 (BSK1) and positively modulates plant resistance. In this study, by a genome-wide phosphorylation analysis, we found that the phosphorylation of BSK1 and OST1 was missing in the rlk902 mutant compared with the wild-type plants, indicating a potential connection between the RLK902-BSK1 module and OST1-mediated stomatal closure. We showed that RLK902 and BSK1 contribute to stomatal immunity, as the stomatal closure induced by the bacterial pathogen Pto DC3000 was impaired in rlk902 and bsk1-1 mutants. Stomatal immunity mediated by RLK902 was dependent on BSK1 phosphorylation at Ser230, a key phosphorylation site for BSK1 functions. Several phosphorylation sites of OST1 were important for RLK902- and BSK1-mediated stomatal immunity. Interestingly, the phosphorylation of Ser171 and Ser175 in OST1 contributed to the stomatal immunity mediated by RLK902 but not by BSK1, while phosphorylation of OST1 at Ser29 and Thr176 residues was critical for BSK1-mediated stomatal immunity. Taken together, these results indicate that RLK902 and BSK1 contribute to disease resistance via OST1-mediated stomatal closure. This work revealed a new function of BSK1 in activating stomatal immunity, and the role of RLK902-BSK1 and OST1 module in regulating pathogen-induced stomatal movement.

Strigolactones (SLs) are plant hormones that regulate diverse developmental processes and environmental responses in plants. It has been discovered that SLs play an important role in regulating plant immune resistance to pathogens but there are currently no reports on their role in the interaction between Nicotiana benthamiana and the tobacco mosaic virus (TMV). In this study, the exogenous application of SLs weakened the resistance of N. benthamiana to TMV, promoting TMV infection, whereas the exogenous application of Tis108, a SL inhibitor, resulted in the opposite effect. Virus-induced gene silencing (VIGS) inhibition of two key SL synthesis enzyme genes, NtCCD7 and NtCCD8, enhanced the resistance of N. benthamiana to TMV. Additionally, we conducted a screening of N. benthamiana related to TMV infection. TMV-infected plants treated with SLs were compared to the control by using RNA-seq. The KEGG enrichment analysis and weighted gene co-expression network analysis (WGCNA) of differentially expressed genes (DEGs) suggested that plant hormone signaling transduction may play a significant role in the SL-TMV-N. benthamiana interactions. This study reveals new functions of SLs in regulating plant immunity and provides a reference for controlling TMV diseases in production.

Climate change presents numerous challenges for agriculture, including frequent events of plant abiotic stresses such as elevated temperatures that lead to heat stress (HS). As the primary driving factor of climate change, HS threatens global food security and biodiversity. In recent years, HS events have negatively impacted plant physiology, reducing plant\'s ability to maintain disease resistance and resulting in lower crop yields. Plants must adapt their priorities toward defense mechanisms to tolerate stress in challenging environments. Furthermore, selective breeding and long-term domestication for higher yields have made crop varieties vulnerable to multiple stressors, making them more susceptible to frequent HS events. Studies on climate change predict that concurrent HS and biotic stresses will become more frequent and severe in the future, potentially occurring simultaneously or sequentially. While most studies have focused on singular stress effects on plant systems to examine how plants respond to specific stresses, the simultaneous occurrence of HS and biotic stresses pose a growing threat to agricultural productivity. Few studies have explored the interactions between HS and plant-biotic interactions. Here, we aim to shed light on the physiological and molecular effects of HS and biotic factor interactions (bacteria, fungi, oomycetes, nematodes, insect pests, pollinators, weedy species, and parasitic plants), as well as their combined impact on crop growth and yields. We also examine recent advances in designing and developing various strategies to address multi-stress scenarios related to HS and biotic factors.