Fonio (Digitaria exilis), an orphan millet crop, is the oldest indigenous crop in West Africa. Although the yield is low due to pre-domestication characteristics, the quick maturation time, drought tolerance, and the ability to thrive on poor soils make fonio a climate-smart crop. Being holobionts, plants evolve in close interaction with microbial partners, which is crucial for plant phenology and fitness. As seeds are the bottleneck of vertically transmitting plant microbiota, we proposed to unravel the seed microbiome of the under-domesticated and resilient crop fonio. Our study investigated the bacterial seed endophyte diversity across 126 sequenced fonio accessions from distinct locations in West Africa. We conducted a correlation study of the structures and functions of the seed-associated microbiomes with the native geo-climate and soil structure data. We also performed Genome-wide association studies (GWAS) to identify genetic loci associated with seed endophyte diversity. We report that fonio millet has diverse heritable seed endophytic taxa. We analyzed the seed microbiomes of 126 fonio accessions and showed that despite the diversity of microbiomes from distinct geographical locations, all fonio genetic groups share a core microbiome. In addition, we observed that native soil composition, geo-climatic factors, and host genotype correlate with the seed microbiomes. GWAS analysis of genetic loci associated with endophyte seed bacterial diversity identified fonio SNPs associated with genes functioning in embryo development and stress/defense response. Analysis of the seed endophyte of the climate-smart crop fonio indicated that despite possessing a heritable core microbiome, native conditions may shape the overall fonio seed microbiomes in different populations. These distinct microbiomes could play important roles in the adaptation of fonio to different environmental conditions. Our study identified the seed microbiome as a potential target for enhancing crop resilience to climate stress in a sustainable way.

ABSTRACT N6-methyladenosine (m6A) is a common and dynamic epitranscriptomic modification in eukaryotic RNAs, affecting stability, splicing, translation, and degradation. Recent technological advancements have revealed the complex nature of m6A modifications, highlighting their importance in plant and animal species. The m6A modification is a reversible process, with “writers” depositing methylation, “erasers” demethylating it, and “reader” proteins recognizing m6A and executing various biological functions. Studying the relationship between m6A methylation and viral infection is crucial. Animal viruses, including retroviruses, RNA viruses, and DNA viruses, often employ the host’s m6A machinery to replicate or avoid immune responses. In plant viruses, host methyltransferases or demethylases can stabilize or degrade viral RNA, depending on the virus-host interaction. Additionally, viral infections can modify the host’s m6A machinery, impacting the viral life cycle. This review examines the role of m6A modifications in plant viral pathogenesis, focussing on RNA viruses infecting crops like alfalfa, turnip, wheat, rice, and potato. Understanding the role of m6A in virus-host interactions can aid in studying plant viral disease development and discovering novel antiviral targets for crop protection. In this review, we summarize current information on m6A in RNA biology, focussing on its function in viral infections and plant-virus interactions.

14-3-3 proteins, ubiquitously present in eukaryotic cells, are regulatory proteins involved in a plethora of cellular processes. In plants, they have been studied in the context of metabolism, development, and stress responses. Recent studies have highlighted the pivotal role of 14-3-3 proteins in regulating plant immunity. The ability of 14-3-3 proteins to modulate immune responses is primarily attributed to their function as interaction hubs, mediating protein-protein interactions and thereby regulating the activity and overall function of their binding partners. Here, we shed light on how 14-3-3 proteins contribute to plant defense mechanisms, the implications of their interactions with components of plant immunity cascades, and the potential for leveraging this knowledge for crop improvement strategies.

Senescence is an important physiological process which directly affects many agronomic traits in plants. Senescence induces chlorophyll degradation, phytohormone changes, cellular structure damage, and altered gene regulation. Although these physiological outputs are well defined, the molecular mechanisms employed are not known. Using dark-induced leaf senescence (DILS) as the experimental system, we investigated the role of N6-methyladenosine (m6A) mRNA methylation during senescence in Arabidopsis (Arabidopsis thaliana). Plants compromised in m6A machinery components like METHYLTRANSFERASE A (mta mutant) and VIRILIZER1 (vir-1 mutant) showed an enhanced DILS phenotype. This was accompanied by compromised chloroplast and photosynthesis performance in mta as well as accumulation of senescence-promoting camalexin and phytohormone jasmonic acid after dark treatment. m6A levels increased during DILS and destabilized senescence-related transcripts thereby preventing premature aging. Due to inefficient decay, senescence-related transcripts like ORESARA1 (ORE1), SENESCENCE-ASSOCIATED GENE 21 (SAG21), NAC-like, activated by AP3/PI (NAP), and NONYELLOWING 1 (NYE1) over-accumulated in mta thereby causing accelerated senescence during DILS. Overall, our data propose that m6A modification is involved in regulating the biological response to senescence in plants, providing targets for engineering stress tolerance of crops.

The use of beneficial microbes to mitigate drought stress tolerance of plants is of great potential albeit little understood. We show here that a root endophytic desert bacterium, Pseudomonas argentinensis sp. SA190, enhances drought stress tolerance in Arabidopsis. Transcriptome and genetic analysis demonstrate that SA190-induced root morphogenesis and gene expression is mediated via the plant abscisic acid (ABA) pathway. Moreover, we demonstrate that SA190 primes the promoters of target genes in an epigenetic manner which is ABA-dependent. Application of the SA190 priming technology on crops is demonstrated for alfalfa in field trials, showing enhanced performance under desert agriculture conditions. In summary, a single beneficial root bacterial strain can help to perform agriculture under drought and water limiting conditions. Synopsis Beneficial root endophyte Pseudomonas argentinensis sp. SA190 confers drought tolerance in plants SA190 modulates the expression of genes under drought stress in an ABA-dependent manner SA190 primes genes via H3K4me3 histone mark enrichment SA190 alters host plant physiology by improving the plant water status SA190 enhances crop performance in open field conditions with limited irrigation

The use of beneficial microbes to mitigate drought stress tolerance of plants is of great potential albeit little understood. We show here that a root endophytic desert bacterium, Pseudomonas argentinensis strain SA190, enhances drought stress tolerance in Arabidopsis. Transcriptome and genetic analysis demonstrate that SA190-induced root morphogenesis and gene expression is mediated via the plant abscisic acid (ABA) pathway. Moreover, we demonstrate that SA190 primes the promoters of target genes in an epigenetic ABA-dependent manner. Application of SA190 priming on crops is demonstrated for alfalfa, showing enhanced performance under drought conditions. In summary, a single beneficial root bacterial strain can help plants to resist drought conditions.

Linker H1 histones play an important role in animal and human pathogenesis, but their function in plant immunity is poorly understood. Here, we analyzed mutants of the three canonical variants of Arabidopsis H1 histones, namely H1.1, H1.2 and H1.3. We observed that double h1.1h1.2 and triple h1.1h1.2h1.3 (3h1) mutants were resistant to Pseudomonas syringae and Botrytis cinerea infections. Transcriptome analysis of 3h1 mutant plants showed H1s play a key role in regulating the expression of early and late defense genes upon pathogen challenge. Moreover, 3h1 mutant plants showed enhanced production of reactive oxygen species and activation of mitogen activated protein kinases upon pathogen-associated molecular pattern (PAMP) treatment. However, 3h1 mutant plants were insensitive to priming with flg22, a well-known bacterial PAMP which induces enhanced resistance in WT plants. The defective defense response in 3h1 upon priming was correlated with altered DNA methylation and reduced global H3K56ac levels. Our data place H1 as a molecular gatekeeper in governing dynamic changes in the chromatin landscape of defense genes during plant pathogen interaction.

In plants, the detection of microbe-associated molecular patterns (MAMPs) induces primary innate immunity by the activation of mitogen-activated protein kinases (MAPKs). We show here that the MAMP-activated MAPK MPK6 not only modulates defense through transcriptional regulation but also via the ribosomal protein translation machinery. To understand the effects of MPK6 on ribosomes and their constituent ribosomal proteins (RPs), polysomes, monosomes and the phosphorylation status of the RPs, MAMP-treated WT and mpk6 mutant plants were analysed. MAMP-activation induced rapid changes in RP composition of monosomes, polysomes and in the 60S ribosomal subunit in an MPK6-specific manner. Phosphoproteome analysis showed that MAMP-activation of MPK6 regulates the phosphorylation status of the P-stalk ribosomal proteins by phosphorylation of RPP0 and the concomitant dephosphorylation of RPP1 and RPP2. These events coincide with a significant decrease in the abundance of ribosome-bound RPP0s, RPP1s and RPP3s in polysomes. The P-stalk is essential in regulating protein translation by recruiting elongation factors. Accordingly, we found that RPP0C mutant plants are compromised in basal resistance to Pseudomonas syringae infection. These data suggest that MAMP-induced defense also involves MPK6-induced regulation of P-stalk proteins, highlighting a new role of ribosomal regulation in plant innate immunity.

Posttranscriptional regulation of mRNA mediated by methylation at the N6 position of adenine (N6-methyladenosine [m6A]) has profound effects on transcriptome regulation in plants. Focused studies across eukaryotes offer glimpses into the processes governed by m6A throughout developmental and disease states. However, we lack an understanding of the dynamics and the regulatory potential of m6A during biotic stress in plants. Here, we provide a comprehensive look into the effects of m6A on both the short-term and long-term responses to pathogen signaling in Arabidopsis (Arabidopsis thaliana). We demonstrate that m6A-deficient plants are more resistant to bacterial and fungal pathogen infections and have altered immune responses. Furthermore, m6A deposition is specifically coordinated on transcripts involved in defense and immunity prior to and proceeding the pathogen signal flagellin. Consequently, the dynamic modulation of m6A on specific stress-responsive transcripts is correlated with changes in abundance and cleavage of these transcripts. Overall, we show that the m6A methylome is regulated prior to and during simulated and active pathogen stress and functions in the coordination and balancing of normal growth and pathogen responses.

The INDETERMINATE DOMAIN (IDD) family belongs to a group of plant-specific transcription factors that coordinates plant growth/development and immunity. However, the function and mode of action of IDDs during abiotic stress, such as salt, are poorly understood. We used idd4 transgenic lines and screened them under salt stress to find the involvement of IDD4 in salinity stress tolerance The genetic disruption of IDD4 increases salt-tolerance, characterized by sustained plant growth, improved Na+/K+ ratio, and decreased stomatal density/aperture. Yet, IDD4 overexpressing plants were hypersensitive to salt-stress with an increase in stomatal density and pore size. Transcriptomic and ChIP-seq analyses revealed that IDD4 directly controls an important set of genes involved in abiotic stress/salinity responses. Interestingly, using anti-IDD4-pS73 antibody we discovered that IDD4 is specifically phosphorylated at serine-73 by MPK6 in vivo under salinity stress. Analysis of plants expressing the phospho-dead and phospho-mimicking IDD4 versions proved that phosphorylation of IDD4 plays a crucial role in plant transcriptional reprogramming of salt-stress genes. Altogether, we show that salt stress adaption involves MPK6 phosphorylation of IDD4 thereby regulating IDD4 DNA-binding and expression of target genes.

In both plants and animals, nucleotide-binding leucine-rich repeat (NLR) immune receptors play critical roles in pathogen recognition and activation of innate immunity. In plants, NLRs recognise pathogen-derived effector proteins and initiate effector-triggered immunity (ETI). However, the molecular mechanisms that link NLR-mediated effector recognition and downstream signalling are not fully understood. By exploiting the well-characterised tomato Prf/Pto NLR resistance complex, we identified the 14-3-3 proteins TFT1 and TFT3 as interacting partners of both the NLR complex and the protein kinase MAPKKKα. Moreover, we identified the helper NRC proteins (NLR-required for cell death) as integral components of the Prf /Pto NLR recognition complex. Notably our studies revealed that TFTs and NRCs interact with distinct modules of the NLR complex and, following effector recognition, dissociate facilitating downstream signalling. Thus, our data provide a mechanistic link between activation of immune receptors and initiation of downstream signalling cascades.

Plants have developed a robust transcription machinery to combat potential pathogenic organisms. One of the hallmarks of early immune responses is the activation of the WRKY transcription factors post infection. Specific WRKYs proteins from Arabidopsis are known substrates of MAPK pathway to mediate the flg22 elicited early immunity. In the current study, using the Golden Gate cloning strategy, we aim to clone the entire WRKY transcription factor family from Oryza sativa ssp. indica consisting of more than 100 members and study their MAPK interaction and subsequent role in PTI. Using a reporter LUC assay in protoplasts we investigated the early defense responses in a few interesting OsWRKY candidates. Interestingly, we observed stringent regulation of WRKY expression in cells and their transcriptional expression only under specific stress responses. The phenomenon of gene expression regulation by intron retention (IR) was prevalently observed in rice WRKY transcripts. We could show the role of WRKY8, 24, and 77 in early defense responses. It was observed that WRKY24 enhanced the expression of early defense response marker genes like NHL10 while WRKY8 and WRKY77 supressed their expression. This study highlights the complicated mechanism by which OsWRKYs expression is possibly regulated and the distinctive roles of some individual members in plant immunity. At the same time this study serves as a cautionary warning for plant researchers to be mindful of the intron retention mechanism while cloning OsWRKYs.

Global warming has become a critical challenge to food security, causing severe yield losses of major crops worldwide. Conventional and transgenic breeding strategies to enhance plant thermotolerance are laborious and expensive. Therefore, the use of beneficial microbes could be an alternative approach. Here, we report that the root endophyte Enterobacter sp. SA187 induces thermotolerance in wheat in the laboratory as well as in open-field agriculture. To unravel the molecular mechanisms, we used Arabidopsis thaliana as model plant. SA187 reprogramed the Arabidopsis transcriptome via HSFA2-dependent enhancement of H3K4me3 levels at heat stress memory gene loci. Unlike thermopriming, SA187-induced thermotolerance is mediated by ethylene signaling via the transcription factor EIN3. In contrast to the transient chromatin modification by thermopriming, SA187 induces constitutive H3K4me3 modification of heat stress memory genes, generating robust thermotolerance in plants. Importantly, microbial community composition of wheat plants in open-field agriculture is not influenced by SA187, indicating that beneficial microbes can be a powerful tool to enhance thermotolerance of crops in a sustainable manner.

Oxylipins constitute a huge class of compounds produced by oxidation of long-chain unsaturated fatty acids either chemically (by radicals such as reactive oxygen species, ROS) or enzymatically (by lipoxygenases, LOX; cyclooxygenases, COX; or cytochrome P450 pathways). This process generates fatty acids peroxides, which can then be further modified in a broad range to epoxy, hydroxy, keto, ether fatty acids, and also hydrolyzed to generate small aldehydes and alcohols. In general, oxylipins are present in almost all living organisms and have a wide range of signaling, metabolic, physiological, and ecological roles depending on the particular organism and on their structure. In plants, oxylipins have been extensively studied over the past 35 years. However, these studies have focused mainly on the jasmonates and so-called green leaves volatiles. The function of early LOX products (like keto and hydroxy fatty acids) is yet not well understood in plants, where they are mainly analyzed by indirect methods or by GC-MS what requires a laborious sample preparation. Here, we developed and validated a straightforward, precise, accurate, and sensitive method for quantifying oxylipins in plant tissues using HPLC-MS/MS, with a one-step extraction procedure using low amount of plant tissues. We successfully applied this method to quantify the oxylipins in different plant species and Arabidopsis thaliana plants treated with various biotic and abiotic stress conditions.

Strawberry ( Fragaria × ananassa) is an economically important high-value crop that is susceptible to three most devastating pathogens with different lifestyles – a necrotrophic fungus Botrytis cinerea causing grey mold, a hemibiotrophic oomycete Phytophthora cactorum causing crown/root rot, and a biotrophic fungus Podosphaera aphanis causing powdery mildew. Studies on individual plant-pathogen interactions are only sufficient for developing disease resistant strawberry varieties to a particular pathogen. However, each of these pathogens have the potential to co-infect strawberry at a given point of time. Therefore, understanding how these pathogens manipulate strawberry’s defences and how it responds to these pathogens is essential for developing broad-spectrum disease resistant varieties. Here, in the diploid model Fragaria vesca , we performed comparative transcriptome analysis between each of these pathogen infections to identify 501 Co mmon Re sponsive (CoRe) genes targeted against these pathogens. Furthermore, about 80% of these CoRe set are upregulated upon infection by all three pathogens indicating a similar transcriptional response of F. vesca independent of pathogen’s lifestyle. These upregulated CoRe set include genes from well-known defence responsive pathways such as calcium and MAP kinase signalling, WRKY transcription factors, pathogenesis-related allergen genes and hormone and terpene biosynthetic genes. These novel insights into F. vesca ’s defences might serve as a basis for engineering plants with broad spectrum resistance.

Global warming has become a critical challenge to food safety, causing severe yield losses of major crops worldwide. Heat acclimation empowers plants to survive under extreme temperature conditions but the potential of beneficial microbes to make plants thermotolerant has not been considered so far. Here, we report that the endophytic bacterium Enterobacter sp. SA187 induces heat tolerance in Arabidopsis thaliana by reprogramming the plant transcriptome to a similar extent as acclimation. Acclimation induces priming of heat stress memory genes such as APX2 and HSP18.2 via the transcription factors HSFA1A, B, D, and E and the downstream master regulator HSFA2. hsfa1a,b,d,e and hsfa2 mutants compromised both acclimation and bacterial priming through the same pathway of HSF transcription factors. However, while acclimation transiently modifies H3K4me3 levels at heat stress memory gene loci, SA187 induces the constitutive priming of these loci. In summary, we demonstrate the molecular mechanism by which SA187 imparts thermotolerance in A. thaliana , suggesting that beneficial microbes might be a promising way to enhance crop production under global warming conditions.

The major virulence strategy of the phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000 is to secrete effector proteins into tomato cells to target the immune machinery. AvrPto and AvrPtoB are two such effectors from P. syringae, which disable a range of kinases in tomato (Solanum lycopersicum). Both effectors target surface-localized receptor-kinases to avoid bacterial recognition. In turn, tomato has evolved an intracellular effector-recognition complex composed of the NB-LRR protein Prf and the Pto kinase. AvrPto is an inhibitor of Pto kinase activity but, paradoxically, this kinase activity is a prerequisite for defence activation by AvrPto. Using biochemical approaches, we show that disruption of the Pto P+1 loop stimulates phosphorylation in trans, which is possible because the Pto/Prf complex is oligomeric. Following P+1 loop disruption and transphosphorylation, the Pto/Prf complex dissociates, leading to downstream signalling through mitogen-activated protein kinases (MAPKs). Hence, the Pto/\Prf complex is a sophisticated molecular trap for effectors and provides an excellent model to study the mechanism of MAPK activation. In the current study, we sought to investigate the mechanism of MAPK activation after Pto/\Prf recognition of the AvrPto/AvrPtoB effectors.

Arabidopsis PR1 is a salicylic acid (SA) inducible marker gene for systemic acquired resistance (SAR). However, the regulation of PR1 in plants is poorly understood. In this study, we showed that AtWRKY50 transcription factor binds to two promoter elements of PR1 via its DNA binding domain. Interestingly, the DNA-binding sites for AtWRKY50 deviate significantly from the consensus WRKY binding W-box. The binding sites are located in close proximity to the binding sites for TGA transcription factors. Transactivation experiments in Arabidopsis protoplasts derived from wild type, npr1-1 and tga256 mutant plants indicated that AtWRKY50 alone was able to induce expression of a PR1::β-glucuronidase (GUS) reporter gene, independent of TGAs or NPR1. However, co-expression of TGA2 or TGA5 with AtWRKY50 synergistically enhanced expression to high levels. Yeast-2-hybrid assays and bimolecular fluorescence complementation (BiFC) experiments revealed that AtWRKY50 could interact with TGA2 and TGA5. Using electrophoretic mobility shift assays (EMSA) it was established that AtWRKY50 and TGA2 or TGA5 simultaneously bind to the PR1 promoter. Taken together, these results support a role of AtWRKY50 in SA-induced expression of PR1. Highlights: AtWRKY50 specifically binds to LS10 region of PR1 promoter and interacts with TGAs to synergistically activate PR1 expression.