Helicases clear hurdles during plant defense protein translation.

Plants undergo translational reprogramming when they are under attack by pathogens. Xiang et al. recently revealed that plant helicases induced by pathogen recognition unwind RNA hairpins upstream of the main open reading frames (mORFs), thus allowing ribosomes to bypass the upstream ORFs (uORFs) and translate downstream defense proteins, a mechanism that is also found in mammals.

A vigilant gliding bird protects plants.

The plant hormone salicylic acid (SA) receptor NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1) plays a critical role for plant defense against biotrophic and hemi-biotrophic pathogens. In a milestone paper, Kumar, Zavaliev, Wu et al. unraveled the structural basis for the assembly of an enhanceosome by NPR1 in activating the expression of plant defense genes.

PBS3: a versatile player in and beyond salicylic acid biosynthesis in Arabidopsis.

AVRPPHB SUSCEPTIBLE 3 (PBS3) belongs to the GH3 family of acyl acid amido synthetases, which conjugates amino acids to diverse acyl acid substrates. Recent studies demonstrate that PBS3 in Arabidopsis plays a key role in the biosynthesis of plant defense hormone salicylic acid (SA) by catalyzing the conjugation of glutamate to isochorismate to form isochorismate-9-glutamate, which is then used to produce SA through spontaneous decay or ENHANCED PSEUDOMONAS SUSCEPTIBILITY (EPS1) catalysis. Consistent with its function as an essential enzyme for SA biosynthesis, PBS3 is well known to be a positive regulator of plant immunity in Arabidopsis. Additionally, PBS3 is also involved in the trade-off between abiotic and biotic stress responses in Arabidopsis by suppressing the inhibitory effect of abscisic acid on SA-mediated plant immunity. Besides stress responses, PBS3 also plays a role in plant development. Under long-day conditions, PBS3 influences Arabidopsis flowering time by regulating the expression of flowering regulators FLOWERING LOCUS C and FLOWERING LOCUS T. Taken together, PBS3 functions in the signaling network of plant development and responses to biotic and/or abiotic stresses, but the molecular mechanisms underlying its diverse roles remain obscure.

AI-Based Protein Interaction Screening and Identification (AISID).

In this study, we presented an AISID method extending AlphaFold-Multimer's success in structure prediction towards identifying specific protein interactions with an optimized AISIDscore. The method was tested to identify the binding proteins in 18 human TNFSF (Tumor Necrosis Factor superfamily) members for each of 27 human TNFRSF (TNF receptor superfamily) members. For each TNFRSF member, we ranked the AISIDscore among the 18 TNFSF members. The correct pairing resulted in the highest AISIDscore for 13 out of 24 TNFRSF members which have known interactions with TNFSF members. Out of the 33 correct pairing between TNFSF and TNFRSF members, 28 pairs could be found in the top five (including 25 pairs in the top three) seats in the AISIDscore ranking. Surprisingly, the specific interactions between TNFSF10 (TNF-related apoptosis-inducing ligand, TRAIL) and its decoy receptors DcR1 and DcR2 gave the highest AISIDscore in the list, while the structures of DcR1 and DcR2 are unknown. The data strongly suggests that AlphaFold-Multimer might be a useful computational screening tool to find novel specific protein bindings. This AISID method may have broad applications in protein biochemistry, extending the application of AlphaFold far beyond structure predictions.

The Interplay between Hydrogen Sulfide and Phytohormone Signaling Pathways under Challenging Environments.

Hydrogen sulfide (H2S) serves as an important gaseous signaling molecule that is involved in intra- and intercellular signal transduction in plant-environment interactions. In plants, H2S is formed in sulfate/cysteine reduction pathways. The activation of endogenous H2S and its exogenous application has been found to be highly effective in ameliorating a wide variety of stress conditions in plants. The H2S interferes with the cellular redox regulatory network and prevents the degradation of proteins from oxidative stress via post-translational modifications (PTMs). H2S-mediated persulfidation allows the rapid response of proteins in signaling networks to environmental stimuli. In addition, regulatory crosstalk of H2S with other gaseous signals and plant growth regulators enable the activation of multiple signaling cascades that drive cellular adaptation. In this review, we summarize and discuss the current understanding of the molecular mechanisms of H2S-induced cellular adjustments and the interactions between H2S and various signaling pathways in plants, emphasizing the recent progress in our understanding of the effects of H2S on the PTMs of proteins. We also discuss future directions that would advance our understanding of H2S interactions to ultimately mitigate the impacts of environmental stresses in the plants.

The Xanthomonas type III effector XopAP prevents stomatal closure by interfering with vacuolar acidification.

Plant stomata close rapidly in response to a rise in the plant hormone abscisic acid (ABA) or salicylic acid (SA) and after recognition of pathogen-associated molecular patterns (PAMPs). Stomatal closure is the result of vacuolar convolution, ion efflux, and changes in turgor pressure in guard cells. Phytopathogenic bacteria secrete type III effectors (T3Es) that interfere with plant defense mechanisms, causing severe plant disease symptoms. Here, we show that the virulence and infection of Xanthomonas oryzae pv. oryzicola (Xoc), which is the causal agent of rice bacterial leaf streak disease, drastically increased in transgenic rice (Oryza sativa L.) plants overexpressing the Xoc T3E gene XopAP, which encodes a protein annotated as a lipase. We discovered that XopAP binds to phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2 ), a membrane phospholipid that functions in pH control in lysosomes, membrane dynamics, and protein trafficking. XopAP inhibited the acidification of vacuoles by competing with vacuolar H+ -pyrophosphatase (V-PPase) for binding to PtdIns(3,5)P2 , leading to stomatal opening. Transgenic rice overexpressing XopAP also showed inhibition of stomatal closure when challenged by Xoc infection and treatment with the PAMP flg22. Moreover, XopAP suppressed flg22-induced gene expression, reactive oxygen species burst and callose deposition in host plants, demonstrating that XopAP subverts PAMP-triggered immunity during Xoc infection. Taken together, these findings demonstrate that XopAP overcomes stomatal immunity in plants by binding to lipids.

NPR1 Promotes Its Own and Target Gene Expression in Plant Defense by Recruiting CDK8.

NPR1 (NONEXPRESSER OF PR GENES1) functions as a master regulator of the plant hormone salicylic acid (SA) signaling and plays an essential role in plant immunity. In the nucleus, NPR1 interacts with transcription factors to induce the expression of PR (PATHOGENESIS-RELATED) genes and thereby promote defense responses. However, the underlying molecular mechanism of PR gene activation is poorly understood. Furthermore, despite the importance of NPR1 in plant immunity, the regulation of NPR1 expression has not been extensively studied. Here, we show that SA promotes the interaction of NPR1 with both CDK8 (CYCLIN-DEPENDENT KINASE8) and WRKY18 (WRKY DNA-BINDING PROTEIN18) in Arabidopsis (Arabidopsis thaliana). NPR1 recruits CDK8 and WRKY18 to the NPR1 promoter, facilitating its own expression. Intriguingly, CDK8 and its associated Mediator subunits positively regulate NPR1 and PR1 expression and play a pivotal role in local and systemic immunity. Moreover, CDK8 interacts with WRKY6, WRKY18, and TGA transcription factors and brings RNA polymerase II to NPR1 and PR1 promoters and coding regions to facilitate their expression. Our studies reveal a mechanism in which NPR1 recruits CDK8, WRKY18, and TGA transcription factors along with RNA polymerase II in the presence of SA and thereby facilitates its own and target gene expression for the establishment of plant immunity.

Reprogramming and remodeling: transcriptional and epigenetic regulation of salicylic acid-mediated plant defense.

As a plant hormone, salicylic acid (SA) plays essential roles in plant defense against biotrophic and hemibiotrophic pathogens. Significant progress has been made in understanding the SA biosynthesis pathways and SA-mediated defense signaling networks in the past two decades. Plant defense responses involve rapid and massive transcriptional reprogramming upon the recognition of pathogens. Plant transcription factors and their co-regulators are critical players in establishing a transcription regulatory network and boosting plant immunity. A multitude of transcription factors and epigenetic regulators have been discovered, and their roles in SA-mediated defense responses have been reported. However, our understanding of plant transcriptional networks is still limited. As such, novel genomic tools and bioinformatic techniques will be necessary if we are to fully understand the mechanisms behind plant immunity. Here, we discuss current knowledge, provide an update on the SA biosynthesis pathway, and describe the transcriptional and epigenetic regulation of SA-mediated plant immune responses.

Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA.

Cytosine residues in mammalian DNA occur in five forms: cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The ten-eleven translocation (Tet) dioxygenases convert 5mC to 5hmC, 5fC and 5caC in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The Tet family of dioxygenases is widely distributed across the tree of life, including in the heterolobosean amoeboflagellate Naegleria gruberi. The genome of Naegleria encodes homologues of mammalian DNA methyltransferase and Tet proteins. Here we study biochemically and structurally one of the Naegleria Tet-like proteins (NgTet1), which shares significant sequence conservation (approximately 14% identity or 39% similarity) with mammalian Tet1. Like mammalian Tet proteins, NgTet1 acts on 5mC and generates 5hmC, 5fC and 5caC. The crystal structure of NgTet1 in complex with DNA containing a 5mCpG site revealed that NgTet1 uses a base-flipping mechanism to access 5mC. The DNA is contacted from the minor groove and bent towards the major groove. The flipped 5mC is positioned in the active-site pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5mC. The sequence conservation between NgTet1 and mammalian Tet1, including residues involved in structural integrity and functional significance, suggests structural conservation across phyla.

The Role of a Host-Induced Arginase of Xanthomonas oryzae pv. oryzae in Promoting Virulence on Rice.

The plant bacterial pathogen Xanthomonas oryzae pv. oryzae causes bacterial blight of rice, which is one of the most destructive rice diseases prevalent in Asia and parts of Africa. Despite many years of research, how X. oryzae pv. oryzae causes bacterial blight of rice is still not completely understood. Here, we show that the loss of the rocF gene caused a significant decrease in the virulence of X. oryzae pv. oryzae in the susceptible rice cultivar IR24. Bioinformatics analysis demonstrated that rocF encodes arginase. Quantitative real-time PCR and Western blot assays revealed that rocF expression was significantly induced by rice and arginine. The rocF deletion mutant strain showed elevated sensitivity to hydrogen peroxide, reduced extracellular polysaccharide (EPS) production, and reduced biofilm formation, all of which are important determinants for the full virulence of X. oryzae pv. oryzae, compared with the wild-type strain. Taken together, the results of this study revealed a mechanism by which a bacterial arginase is required for the full virulence of X. oryzae pv. oryzae on rice because of its contribution to tolerance to reactive oxygen species, EPS production, and biofilm formation.

Novel Salicylic Acid Analogs Induce a Potent Defense Response in Arabidopsis.

The master regulator of salicylic acid (SA)-mediated plant defense, NPR1 (NONEXPRESSER OF PR GENES 1) and its paralogs NPR3 and NPR4, act as SA receptors. After the perception of a pathogen, plant cells produce SA in the chloroplast. In the presence of SA, NPR1 protein is reduced from oligomers to monomers, and translocated into the nucleus. There, NPR1 binds to TGA, TCP, and WRKY transcription factors to induce expression of plant defense genes. A list of compounds structurally similar to SA was generated using ChemMine Tools and its Clustering Toolbox. Several of these analogs can induce SA-mediated defense and inhibit growth of Pseudomonas syringae in Arabidopsis. These analogs, when sprayed on Arabidopsis, can induce the accumulation of the master regulator of plant defense NPR1. In a yeast two-hybrid system, these analogs can strengthen the interactions among NPR proteins. We demonstrated that these analogs can induce the expression of the defense marker gene PR1. Furthermore, we hypothesized that these SA analogs could be potent tools against the citrus greening pathogen Candidatus liberibacter spp. In fact, our results suggest that the SA analogs we tested using Arabidopsis may also be effective for inducing a defense response in citrus. Several SA analogs consistently strengthened the interactions between citrus NPR1 and NPR3 proteins in a yeast two-hybrid system. In future assays, we plan to test whether these analogs avoid degradation by SA hydroxylases from plant pathogens. In future assays, we plan to test whether these analogs avoid degradation by SA hydroxylases from plant pathogens.

Heat-Stable Antifungal Factor (HSAF) Biosynthesis in Lysobacter enzymogenes Is Controlled by the Interplay of Two Transcription Factors and a Diffusible Molecule.

Lysobacter enzymogenes is a Gram-negative, environmentally ubiquitous bacterium that produces a secondary metabolite, called heat-stable antifungal factor (HSAF), as an antifungal factor against plant and animal fungal pathogens. 4-Hydroxybenzoic acid (4-HBA) is a newly identified diffusible factor that regulates HSAF synthesis via L. enzymogenes LysR (LysRLe), an LysR-type transcription factor (TF). Here, to identify additional TFs within the 4-HBA regulatory pathway that control HSAF production, we reanalyzed the LenB2-based transcriptomic data, in which LenB2 is the enzyme responsible for 4-HBA production. This survey led to identification of three TFs (Le4806, Le4969, and Le3904). Of them, LarR (Le4806), a member of the MarR family proteins, was identified as a new TF that participated in the 4-HBA-dependent regulation of HSAF production. Our data show the following: (i) that LarR is a downstream component of the 4-HBA regulatory pathway controlling the HSAF level, while LysRLe is the receptor of 4-HBA; (ii) that 4-HBA and LysRLe have opposite regulatory effects on larR transcription whereby larR transcript is negatively modulated by 4-HBA while LysRLe, in contrast, exerts positive transcriptional regulation by directly binding to the larR promoter without being affected by 4-HBA in vitro; (iii) that LarR, similar to LysRLe, can bind to the promoter of the HSAF biosynthetic gene operon, leading to positive regulation of HSAF production; and (iv) that LarR and LysRLe cannot interact and instead control HSAF biosynthesis independently. These results outline a previously uncharacterized mechanism by which biosynthesis of the antibiotic HSAF in L. enzymogenes is modulated by the interplay of 4-HBA, a diffusible molecule, and two different TFs.IMPORTANCE Bacteria use diverse chemical signaling molecules to regulate a wide range of physiological and cellular processes. 4-HBA is an "old" chemical molecule that is produced by diverse bacterial species, but its regulatory function and working mechanism remain largely unknown. We previously found that 4-HBA in L. enzymogenes could serve as a diffusible factor regulating HSAF synthesis via LysRLe Here, we further identified LarR, an MarR family protein, as a second TF that participates in the 4-HBA-dependent regulation of HSAF biosynthesis. Our results dissected how LarR acts as a protein linker to connect 4-HBA and HSAF synthesis, whereby LarR also has cross talk with LysRLe Thus, our findings not only provide fundamental insight regarding how a diffusible molecule (4-HBA) adopts two different types of TFs for coordinating HSAF biosynthesis but also show the use of applied microbiology to increase the yield of the antibiotic HSAF by modification of the 4-HBA regulatory pathway in L. enzymogenes.

NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants.

Salicylic acid (SA) is a plant immune signal produced after pathogen challenge to induce systemic acquired resistance. It is the only major plant hormone for which the receptor has not been firmly identified. Systemic acquired resistance in Arabidopsis requires the transcription cofactor nonexpresser of PR genes 1 (NPR1), the degradation of which acts as a molecular switch. Here we show that the NPR1 paralogues NPR3 and NPR4 are SA receptors that bind SA with different affinities. NPR3 and NPR4 function as adaptors of the Cullin 3 ubiquitin E3 ligase to mediate NPR1 degradation in an SA-regulated manner. Accordingly, the Arabidopsis npr3 npr4 double mutant accumulates higher levels of NPR1, and is insensitive to induction of systemic acquired resistance. Moreover, this mutant is defective in pathogen effector-triggered programmed cell death and immunity. Our study reveals the mechanism of SA perception in determining cell death and survival in response to pathogen challenge.

Insights Into the Roles of Two Genes of the Histidine Biosynthesis Operon in Pathogenicity of Xanthomonas oryzae pv. oryzicola.

Xanthomonas oryzae pv. oryzicola is an X. oryzae pathovar that causes bacterial leaf streak in rice. In this study, we performed functional characterization of a nine-gene his operon in X. oryzae pv. oryzicola. Sequence analysis indicates that this operon is highly conserved in Xanthomonas spp. Auxotrophic assays confirmed that the his operon was involved in histidine biosynthesis. We found that two genes within this operon, trpR and hisB, were required for virulence and bacterial growth in planta. Further research revealed that trpR and hisB play different roles in X. oryzae pv. oryzicola. The trpR acts as a transcriptional repressor and could negatively regulate the expression of hisG, -D, -C, -B, -H, -A, and -F. hisB, which encodes a bifunctional enzyme implicated in histidine biosynthesis, was shown to be required for xanthomonadin production in X. oryzae pv. oryzicola. The disruption of hisB reduced the transcriptional expression of five known shikimate pathway-related genes xanB2, aroE, aroA, aroC, and aroK. We found that the his operon in X. oryzae pv. oryzicola is not involved in hypersensitive response in nonhost tobacco plants. Collectively, our results revealed that two genes in histidine biosynthesis operon play an important role in the pathogenicity of X. oryzae pv. oryzicola Rs105.

RNA-Seq Links the Transcription Factors AINTEGUMENTA and AINTEGUMENTA-LIKE6 to Cell Wall Remodeling and Plant Defense Pathways.

AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) are two related transcription factors in Arabidopsis (Arabidopsis thaliana) that have partially overlapping roles in several aspects of flower development, including floral organ initiation, identity specification, growth, and patterning. To better understand the biological processes regulated by these two transcription factors, we performed RNA sequencing (RNA-Seq) on ant ail6 double mutants. We identified thousands of genes that are differentially expressed in the double mutant compared with the wild type. Analyses of these genes suggest that ANT and AIL6 regulate floral organ initiation and growth through modifications to the cell wall polysaccharide pectin. We found reduced levels of demethylesterified homogalacturonan and altered patterns of auxin accumulation in early stages of ant ail6 flower development. The RNA-Seq experiment also revealed cross-regulation of AIL gene expression at the transcriptional level. The presence of a number of overrepresented Gene Ontology terms related to plant defense in the set of genes differentially expressed in ant ail6 suggest that ANT and AIL6 also regulate plant defense pathways. Furthermore, we found that ant ail6 plants have elevated levels of two defense hormones: salicylic acid and jasmonic acid, and show increased resistance to the bacterial pathogen Pseudomonas syringae These results suggest that ANT and AIL6 regulate biological pathways that are critical for both development and defense.

LesR is a novel upstream regulator that controls downstream Clp expression to modulate antibiotic HSAF biosynthesis and cell aggregation in Lysobacter enzymogenes OH11.

BACKGROUND: Heat-stable antifungal factor (HSAF) is a polycyclic tetramate macrolactam secondary metabolite that exhibits broad-spectrum inhibitory activities against filamentous fungal pathogens. The native yield of this chemical is low. It is also a great challenge to synthesize HSAF artificially, due to its complex structure. Understanding the regulatory mechanism underlying HSAF biosynthesis could provide genetic basis for engineering high HSAF-producing strain. The transcription factor Clp is a global regulator that controls bacterial pathogenicity and the expression of one hundred related genes in the phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc). Diffusible signal factor (DSF) chemical signaling is the only well-characterized upstream regulatory pathway that involves downstream Clp regulation in Xcc. Such a regulatory hierarchy between DSF signaling and Clp is also conserved in the Gram-negative biological control agent Lysobacter enzymogenes, where the DSF signaling system controls antifungal antibiotic HSAF biosynthesis via Clp. RESULTS: Here, using LLysobacter enzymogenes OH11 as a working organism, we examined a novel upstream regulator, LesR, a LuxR solo that controls Clp expression to modulate HSAF biosynthesis as well as cell aggregation. We found that the overexpression of lesR in strain OH11 almost entirely shut down HSAF production and accelerated cell aggregation. These changed phenotypes could be rescued by the introduction of plasmid-borne clp in the lesR overexpression background. Consistent with findings, we further found that overexpression of lesR led to a decrease in the Clp level. CONCLUSIONS: These results collectively have shown that LesR could exert its function, i.e., HSAF biosynthesis, via downstream Clp. These findings were subsequently validated by a comparative transcriptome analysis, where the regulatory action of LesR was found to largely overlap with that of Clp. Therefore, in addition to the well-known DSF signaling system, the present study reveals that LesR functions as a new upstream regulatory factor of Clp in L. enzymogenes. The key factor was important for the production of HSAF. The strains with high HSAF yield can presumably be constructed by deletion of the negative regulators or overexpression of the positive regulators by genetic engineering.

Effector-triggered and self-regulated plant resistance to insects.

Despite many years of research, the molecular mechanisms underlying the activation and regulation of host plant resistance (HPR) to insects remain elusive. Recently, Guo et al. reported that a nucleotide-binding leucine-rich repeat NLR protein activates HPR through direct recognition of an insect effector and that autophagy-mediated degradation of this effector negatively regulates HPR.

Fortifying nematode resistance through susceptibility gene inactivation.

The predominant genetic defense mechanism against soybean cyst nematode (SCN) in 95% of the North America market is under threat by virulent SCN populations. Usovsky et al. identified GmSNAP02 as an SCN susceptibility gene through fine-mapping of unique bi-parental populations. Loss-of-function of GmSNAP02 confers enhanced resistance to more virulent SCN.

Join the green team: Inducers of plant immunity in the plant disease sustainable control toolbox.

BACKGROUND: Crops are constantly attacked by various pathogens. These pathogenic microorganisms, such as fungi, oomycetes, bacteria, viruses, and nematodes, threaten global food security by causing detrimental crop diseases that generate tremendous quality and yield losses worldwide. Chemical pesticides have undoubtedly reduced crop damage; however, in addition to increasing the cost of agricultural production, the extensive use of chemical pesticides comes with environmental and social costs. Therefore, it is necessary to vigorously develop sustainable disease prevention and control strategies to promote the transition from traditional chemical control to modern green technologies. Plants possess sophisticated and efficient defense mechanisms against a wide range of pathogens naturally. Immune induction technology based on plant immunity inducers can prime plant defense mechanisms and greatly decrease the occurrence and severity of plant diseases. Reducing the use of agrochemicals is an effective way to minimize environmental pollution and promote agricultural safety. AIM OF REVIEW: The purpose of this workis to offer valuable insights into the current understanding and future research perspectives of plant immunity inducers and their uses in plant disease control, ecological and environmental protection, and sustainable development of agriculture. KEY SCIENTIFIC CONCEPTS OF REVIEW: In this work, we have introduced the concepts of sustainable and environment-friendly concepts of green disease prevention and control technologies based on plant immunity inducers. This article comprehensively summarizes these recent advances, emphasizes the importance of sustainable disease prevention and control technologies for food security, and highlights the diverse functions of plant immunity inducers-mediated disease resistance. The challenges encountered in the potential applications of plant immunity inducers and future research orientation are also discussed.