PHYTOALEXIN DEFICIENT4 Research Articles

A comprehensive review of soybean RNL and TIR domain proteins.

Both prokaryotic and eukaryotic organisms use the nucleotide-binding domain/leucine-rich repeat (NBD/LRR)-triggered immunity (NLR-triggered immunity) signaling pathway to defend against pathogens. Plant NLRs are intracellular immune receptors that can bind to effector proteins secreted by pathogens. Dicotyledonous plants express a type of NLR known as TIR domain-containing NLRs (TNLs). TIR domains are enzymes that catalyze the production of small molecules that are essential for immune signaling and lead to plant cell death. The activation of downstream TNL signaling components, such as enhanced disease susceptibility 1 (EDS1), phytoalexin deficient 4 (PAD4), and senescence-associated gene 101 (SAG101), is facilitated by these small molecules. Helper NLRs (hNLRs) and the EDS1-PAD4/SAG101 complex associate after activation, causing the hNLRs to oligomerize, translocate to the plasma membrane (PM), and produce cation-selective channels. According to a recent theory, cations enter cells through pores created by oligomeric hNLRs and trigger cell death. Occasionally, TNLs can self-associate to create higher-order oligomers. Here, we categorized soybean TNLs based on the protein domains that they possess. We believe that TNLs may help soybean plants effectively fight pathogens by acting as a source of genetic resistance. In summary, the purpose of this review is to elucidate the range of TNLs that are expressed in soybean.

Subtilisin-like proteases from Fusarium graminearum induce plant cell death and contribute to virulence.

Fusarium head blight (FHB), caused by Fusarium graminearum, causes huge annual economic losses in cereal production. To successfully colonize host plants, pathogens secrete hundreds of effectors that interfere with plant immunity and facilitate infection. However, the roles of most secreted effectors of F. graminearum in pathogenesis remain unclear. We analyzed the secreted proteins of F. graminearum and identified 255 candidate effector proteins by liquid chromatography-mass spectrometry (LC-MS). Five subtilisin-like family proteases (FgSLPs) were identified that can induce cell death in Nicotiana benthamiana leaves. Further experiments showed that these FgSLPs induced cell death in cotton (Gossypium barbadense) and Arabidopsis (Arabidopsis thaliana). A signal peptide and light were not essential for the cell death-inducing activity of FgSLPs. The I9 inhibitor domain and the entire C-terminus of FgSLPs were indispensable for their self-processing and cell death-inducing activity. FgSLP-induced cell death occurred independent of the plant signal transduction components BRI-ASSOCIATED KINASE 1 (BAK1), SUPPRESSOR OF BIR1 1 (SOBIR1), ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), and PHYTOALEXIN DEFICIENT 4 (PAD4). Reduced virulence was observed when FgSLP1 and FgSLP2 were simultaneously knocked out. This study reveals a class of secreted toxic proteins essential for F. graminearum virulence.

METACASPASE8 (MC8) Is a Crucial Protein in the LSD1-Dependent Cell Death Pathway in Response to Ultraviolet Stress.

LESION-SIMULATING DISEASE1 (LSD1) is one of the well-known cell death regulatory proteins in Arabidopsis thaliana. The lsd1 mutant exhibits runaway cell death (RCD) in response to various biotic and abiotic stresses. The phenotype of the lsd1 mutant strongly depends on two other proteins, ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and PHYTOALEXIN-DEFICIENT 4 (PAD4) as well as on the synthesis/metabolism/signaling of salicylic acid (SA) and reactive oxygen species (ROS). However, the most interesting aspect of the lsd1 mutant is its conditional-dependent RCD phenotype, and thus, the defined role and function of LSD1 in the suppression of EDS1 and PAD4 in controlled laboratory conditions is different in comparison to a multivariable field environment. Analysis of the lsd1 mutant transcriptome in ambient laboratory and field conditions indicated that there were some candidate genes and proteins that might be involved in the regulation of the lsd1 conditional-dependent RCD phenotype. One of them is METACASPASE 8 (AT1G16420). This type II metacaspase was described as a cell death-positive regulator induced by UV-C irradiation and ROS accumulation. In the double mc8/lsd1 mutant, we discovered reversion of the lsd1 RCD phenotype in response to UV radiation applied in controlled laboratory conditions. This cell death deregulation observed in the lsd1 mutant was reverted like in double mutants of lsd1/eds1 and lsd1/pad4. To summarize, in this work, we demonstrated that MC8 is positively involved in EDS1 and PAD4 conditional-dependent regulation of cell death when LSD1 function is suppressed in Arabidopsis thaliana. Thus, we identified a new protein compound of the conditional LSD1-EDS1-PAD4 regulatory hub. We proposed a working model of MC8 involvement in the regulation of cell death and we postulated that MC8 is a crucial protein in this regulatory pathway.

WRR4B contributes to a broad-spectrum disease resistance against powdery mildew in Arabidopsis.

Oidium heveae HN1106, a powdery mildew (PM) that infects rubber trees, has been found to trigger disease resistance in Arabidopsis thaliana through ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1)-, PHYTOALEXIN DEFICIENT 4 (PAD4)- and salicylic acid (SA)-mediated signalling pathways. In this study, a typical TOLL-INTERLEUKIN 1 RECEPTOR, NUCLEOTIDE-BINDING, LEUCINE-RICH REPEAT (TIR-NB-LRR)-encoding gene, WHITE RUST RESISTANCE 4 (WRR4B), was identified to be required for the resistance against O. heveae in Arabidopsis. The expression of WRR4B was upregulated by O. heveae inoculation, and WRR4B positively regulated the expression of genes involved in SA biosynthesis, such as EDS1, PAD4, ICS1 (ISOCHORISMATE SYNTHASE 1), SARD1 (SYSTEMIC-ACQUIRED RESISTANCE DEFICIENT 1) and CBP60g (CALMODULIN-BINDING PROTEIN 60 G). Furthermore, WRR4B triggered self-amplification, suggesting that WRR4B mediated plant resistance through taking part in the SA-based positive feedback loop. In addition, WRR4B induced an EDS1-dependent hypersensitive response in Nicotiana benthamiana and contributed to disease resistance against three other PM species: Podosphaera xanthii, Erysiphe quercicola and Erysiphe neolycopersici, indicating that WRR4B is a broad-spectrum disease resistance gene against PMs.

Arabidopsis PHYTOALEXIN DEFICIENT 4 promotes the maturation and nuclear accumulation of immune-related cysteine protease RD19.

Arabidopsis PHYTOALEXIN DEFICIENT 4 (PAD4) has an essential role in pathogen resistance as a heterodimer with ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1). Here we investigated an additional PAD4 role in which it associates with and promotes the maturation of the immune-related cysteine protease RESPONSIVE TO DEHYDRATION 19 (RD19). We found that RD19 and its paralog RD19c promoted EDS1- and PAD4-mediated effector-triggered immunity to an avirulent Pseudomonas syringae strain, DC3000, expressing the effector AvrRps4 and basal immunity against the fungal pathogen Golovinomyces cichoracearum. Overexpression of RD19, but not RD19 protease-inactive catalytic mutants, in Arabidopsis transgenic lines caused EDS1- and PAD4-dependent autoimmunity and enhanced pathogen resistance. In these lines, RD19 maturation to a pro-form required its catalytic residues, suggesting that RD19 undergoes auto-processing. In transient assays, PAD4 interacted preferentially with the RD19 pro-protease and promoted its nuclear accumulation in leaf cells. Our results lead us to propose a model for PAD4-stimulated defense potentiation. PAD4 promotes maturation and nuclear accumulation of processed RD19, and RD19 then stimulates EDS1-PAD4 dimer activity to confer pathogen resistance. This study highlights potentially important additional PAD4 functions that eventually converge on canonical EDS1-PAD4 dimer signaling in plant immunity.

The NLR immune receptor ADR1 and lipase-like proteins EDS1 and PAD4 mediate stomatal immunity in Nicotiana benthamiana and Arabidopsis.

In the presence of pathogenic bacteria, plants close their stomata to prevent pathogen entry. Intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogenic effectors and activate effector-triggered immune responses. However, the regulatory and molecular mechanisms of stomatal immunity involving NLR immune receptors are unknown. Here, we show that the Nicotiana benthamiana RPW8-NLR central immune receptor ACTIVATED DISEASE RESISTANCE 1 (NbADR1), together with the key immune proteins ENHANCED DISEASE SUSCEPTIBILITY 1 (NbEDS1) and PHYTOALEXIN DEFICIENT 4 (NbPAD4), plays an essential role in bacterial pathogen- and flg22-induced stomatal immunity by regulating the expression of salicylic acid (SA) and abscisic acid (ABA) biosynthesis or response-related genes. NbADR1 recruits NbEDS1 and NbPAD4 in stomata to form a stomatal immune response complex. The transcription factor NbWRKY40e, in association with NbEDS1 and NbPAD4, modulates the expression of SA and ABA biosynthesis or response-related genes to influence stomatal immunity. NbADR1, NbEDS1, and NbPAD4 are required for the pathogen infection-enhanced binding of NbWRKY40e to the ISOCHORISMATE SYNTHASE 1 promoter. Moreover, the ADR1-EDS1-PAD4 module regulates stomatal immunity in Arabidopsis (Arabidopsis thaliana). Collectively, our findings show the pivotal role of the core intracellular immune receptor module ADR1-EDS1-PAD4 in stomatal immunity, which enables plants to limit pathogen entry.

Inhibition ofNPR1 Leads to Shoot Growth Improvement under Low-Calcium Conditions inArabidopsis.

Under low-Ca conditions, plants accumulate salicylic acid (SA) and induce SA-responsive genes. However, the relationship between SA and low-Ca tolerance remains unclear. Here, we demonstrated that the inhibition or suppression of nonexpressor of pathogenesis-related 1 (NPR1) activity, a major regulator of the SA signaling pathway in the defense response, improves shoot growth under low-Ca conditions. Furthermore, mutations in phytoalexin-deficient 4 (PAD4) or enhanced disease susceptibility 1 (EDS1), which are upstream regulators of NPR1, improved shoot growth under low-Ca conditions, suggesting that NPR1 suppressed growth under low-Ca conditions. In contrast, growth of SA induction-deficient 2-2 (sid2-2), which is an SA-deficient mutant, was sensitive to low Ca levels, suggesting that SA accumulation by SID2 was not related to growth inhibition under low-Ca conditions. Additionally, npr1-1 showed low-Ca tolerance, and the application of tenoxicam-an inhibitor of the NPR1-mediated activation of gene expression-also improved shoot growth under low Ca conditions. The low-Ca tolerance of double mutants pad4-1, npr1-1 and eds1-22 npr1-1 was similar to that of the single mutants, suggesting that PAD4 and EDS1 are involved in the same genetic pathway in suppressing growth under low-Ca conditions as NPR1. Cell death and low-Ca tolerance did not correlate among the mutants, suggesting that growth improvement in the mutants was not due to cell death inhibition. In conclusion, we revealed that NPR1 suppresses plant growth under low-Ca conditions and that the other SA-related genes influence plant growth and cell death.

Biotechnological Potential of the Stress Response and Plant Cell Death Regulators Proteins in the Biofuel Industry.

Production of biofuel from lignocellulosic biomass is relatively low due to the limited knowledge about natural cell wall loosening and cellulolytic processes in plants. Industrial separation of cellulose fiber mass from lignin, its saccharification and alcoholic fermentation is still cost-ineffective and environmentally unfriendly. Assuming that the green transformation is inevitable and that new sources of raw materials for biofuels are needed, we decided to study cell death-a natural process occurring in plants in the context of reducing the recalcitrance of lignocellulose for the production of second-generation bioethanol. "Members of the enzyme families responsible for lysigenous aerenchyma formation were identified during the root hypoxia stress in Arabidopsis thaliana cell death mutants. The cell death regulatory genes, LESION SIMULATING DISEASE 1 (LSD1), PHYTOALEXIN DEFICIENT 4 (PAD4) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) conditionally regulate the cell wall when suppressed in transgenic aspen. During four years of growth in the field, the following effects were observed: lignin content was reduced, the cellulose fiber polymerization degree increased and the growth itself was unaffected. The wood of transgenic trees was more efficient as a substrate for saccharification, alcoholic fermentation and bioethanol production. The presented results may trigger the development of novel biotechnologies in the biofuel industry.

A splicing variant of EDS1 from Vitis vinifera forms homodimers but no heterodimers with PAD4.

Enhanced Disease Susceptibility 1 (EDS1), a key component of microbe-triggered immunity and effector-triggered immunity in most higher plants, forms functional heterodimeric complexes with its homologs Phytoalexin Deficient 4 (PAD4) or Senescence-associated Gene 101 (SAG101). Here, the crystal structure of VvEDS1Nterm , the N-terminal domain of EDS1 from Vitis vinifera, is reported, representing the first structure of an EDS1 entity beyond the model plant Arabidopsis thaliana. VvEDS1Nterm has an α/β-hydrolase fold, is similar to the N-terminal domain of A. thaliana EDS1 and forms stable homodimers in solution as well as in crystals. These VvEDS1Nterm homodimers are spatially incompatible with heterodimers with PAD4 or SAG101, they explain why VvEDS1Nterm does not interact with V. vinifera PAD4 according to gel filtration, and they serve as a guide to develop a plausible, albeit experimentally not verified model of full-length EDS1. VvEDS1Nterm is a splicing variant comprising two of three exons of the VvEDS1 gene. It originates from a naturally occurring mRNA, in which the first of two introns was removed while the second one containing a stop codon close to the exon/intron border was retained. This is a potential case of intron retention and the first report of this phenomenon in the context of EDS1. Its biological significance has not yet been clarified, nor has the question if a VvEDS1Nterm protein with a specific function can occur under physiological conditions.

Identification of HIR, EDS1 and PAD4 Genes Reveals Differences between Coffea Species That May Impact Disease Resistance

Coffee, a widely consumed important agricultural product, is mainly produced from two species, Coffea arabica (Arabica coffee) and C. canephora (Robusta coffee). Timor Hybrid (HDT) is a population resulting from a natural cross between C. arabica and C. canephora. HDT derivatives have a high spectrum of resistance to different races of Hemileia vastatrix (Hv), the causal agent of coffee leaf rust. A RNAseq database, obtained from HDT832/2 leaves inoculated with Hv (Host Resistance) and Uromyces vignae (Uv, Nonhost Resistance), showed the presence of genes implicated in the hypersensitive response and salicylic acid pathway. Hypersensitive Induced Reaction (HIR) gene family, Enhanced Disease Susceptibility1 gene (EDS1), and Phytoalexin Deficient 4 (PAD4) gene are involved in host and nonhost resistance. Relative expression calculated by RT-qPCR was used to confirm and expand the transcriptome analysis. HDTHIR4, HDTEDS1, and HDTPAD4 showed the highest upregulation in response to Hv and Uv inoculation, confirming a similar trend in host and nonhost resistance in HDT. HIR and EDS1/PAD4 gene families were characterized for the first time in the three available Coffea genomes. HIR genes were quite conserved between Coffea species. Surprisingly, EDS1 and PAD4 genes revealed major differences in gene structure. The PAD4 predicted protein from C. arabica does not include both conserved domains of the EDS1/PAD4 family, and the EDS1 putative protein from C. canephora includes a formin domain unusual in the same protein family. The variability shown by EDS1/PAD4 gene family may impact the disease resistance response of Coffea species, which can be surveyed for the gene sequences that will produce a more resistant phenotype.

Autophagy and multivesicular body pathways cooperate to protect sulfur assimilation and chloroplast functions.

Autophagy and multivesicular bodies (MVBs) represent 2 closely related lysosomal/vacuolar degradation pathways. In Arabidopsis (Arabidopsis thaliana), autophagy is stress-induced, with deficiency in autophagy causing strong defects in stress responses but limited effects on growth. LYST-INTERACTING PROTEIN 5 (LIP5) is a key regulator of stress-induced MVB biogenesis, and mutation of LIP5 also strongly compromises stress responses with little effect on growth in Arabidopsis. To determine the functional interactions of these 2 pathways in Arabidopsis, we generated mutations in both the LIP5 and AUTOPHAGY-RELATED PROTEIN (ATG) genes. atg5/lip5 and atg7/lip5 double mutants displayed strong synergistic phenotypes in fitness characterized by stunted growth, early senescence, reduced survival, and greatly diminished seed production under normal growth conditions. Transcriptome and metabolite analysis revealed that chloroplast sulfate assimilation was specifically downregulated at early seedling stages in the atg7/lip5 double mutant prior to the onset of visible phenotypes. Overexpression of adenosine 5'-phosphosulfate reductase 1, a key enzyme in sulfate assimilation, substantially improved the growth and fitness of the atg7/lip5 double mutant. Comparative multi-omic analysis further revealed that the atg7/lip5 double mutant was strongly compromised in other chloroplast functions including photosynthesis and primary carbon metabolism. Premature senescence and reduced survival of atg/lip5 double mutants were associated with increased accumulation of reactive oxygen species and overactivation of stress-associated programs. Blocking PHYTOALEXIN DEFICIENT 4 and salicylic acid signaling prevented early senescence and death of the atg7/lip5 double mutant. Thus, stress-responsive autophagy and MVB pathways play an important cooperative role in protecting essential chloroplast functions including sulfur assimilation under normal growth conditions to suppress salicylic-acid-dependent premature cell-death and promote plant growth and fitness.

Divergent Roles of CNGC2 and CNGC4 in the Regulation of Disease Resistance, Plant Growth and Heat Tolerance in Arabidopsis

Arabidopsis cyclic nucleotide-gated channels (CNGC) 2 and 4 are shown to negatively regulate disease resistance and heat tolerance and to positively regulate plant growth. Whether or not their functions in these processes are interdependent is largely unknown. Here, using the mutation of phytoalexin deficient 4 (PAD4) to inhibit the enhanced defense response and programmed cell death (PCD), we assessed the contribution of the altered defense response to the heat tolerance and plant growth in the cngc2 and cngc4 single and double mutants. The pad4 mutation reverted the enhanced disease resistance of the cngc2 and cngc4 mutants at the normal temperature (22 °C) but not at the elevated temperature (28 °C). The pad4 mutation slightly alleviated the dwarfism of the cngc2 and cngc4 mutants at 22 °C but not at 28 °C, indicating a small contribution from the defense response to plant growth regulation. The pad4 mutation also reduced the enhanced heat tolerance in the cngc mutants, suggesting an involvement of PCD in heat tolerance. In addition, a higher heat tolerance was correlated with more opened stomata under heat treatment among the wild type and mutants of the PAD4, CNGC2 and CNGC4 genes. In sum, this study suggests that the regulation of heat tolerance and plant growth by CNGC2 and CNGC4 is almost independent of their regulation of disease resistance. It also reveals a PAD4-dependent role of CNGC2 and CNGC4 in stomatal aperture regulation and heat tolerance.

TIR-catalyzed ADP-ribosylation reactions produce signaling molecules for plant immunity.

Plant pathogen-activated immune signaling by nucleotide-binding leucine-rich repeat (NLR) receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain converges on Enhanced Disease Susceptibility 1 (EDS1) and its direct partners, Phytoalexin Deficient 4 (PAD4) or Senescence-Associated Gene 101 (SAG101). TIR-encoded nicotinamide adenine dinucleotide hydrolase (NADase) produces signaling molecules to promote exclusive EDS1-PAD4 and EDS1-SAG101 interactions with helper NLR subclasses. In this work, we show that TIR-containing proteins catalyze adenosine diphosphate (ADP)-ribosylation of adenosine triphosphate (ATP) and ADP ribose (ADPR) through ADPR polymerase-like and NADase activity, forming ADP-ribosylated ATP (ADPr-ATP) and ADPr-ADPR (di-ADPR), respectively. Specific binding of ADPr-ATP or di-ADPR allosterically promotes EDS1-SAG101 interaction with helper NLR N requirement gene 1A (NRG1A) in vitro and in planta. Our data reveal an enzymatic activity of TIRs that enables specific activation of the EDS1-SAG101-NRG1 immunity branch.

The Two Classes of Ceramide Synthases Play Different Roles in Plant Immunity and Cell Death.

Ceramide synthases (CSs) produce ceramides from long-chain bases (LCBs). However, how CSs regulate immunity and cell death in Arabidopsis thaliana remains unclear. Here, we decipher the roles of two classes of CS, CSI (LAG1 HOMOLOG 2, LOH2) and CSII (LOH1/3), in these processes. The loh1-2 and loh1-1 loh3-1 mutants were resistant to the bacterial pathogen Pseudomonas syringae pv maculicola (Psm) DG3 and exhibited programmed cell death (PCD), along with increased LCBs and ceramides, at later stages. In loh1-2, the Psm resistance, PCD, and sphingolipid accumulation were mostly suppressed by inactivation of the lipase-like proteins ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and PHYTOALEXIN DEFICIENT 4 (PAD4), and partly suppressed by loss of SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2). The LOH1 inhibitor fumonisin B1 (FB1) triggered EDS1/PAD4-independent LCB accumulation, and EDS1/PAD4-dependent cell death, resistance to Psm, and C16 Cer accumulation. Loss of LOH2 enhances FB1-, and sphinganine-induced PCD, indicating that CSI negatively regulates the signaling triggered by CSII inhibition. Like Cer, LCBs mediate cell death and immunity signaling, partly through the EDS1/PAD4 pathway. Our results show that the two classes of ceramide synthases differentially regulate EDS1/PAD4-dependent PCD and immunity via subtle control of LCBs and Cers in Arabidopsis.

GIGANTEA regulates PAD4 transcription to promote pathogen defense against Hyaloperonospora arabidopsidis in Arabidopsis thaliana

ABSTRACT Plants have evolved a network of complex signaling pathways that allow them to cope with the fluctuations of internal and external environmental cues. GIGANTEA (GI), a well-known, highly conserved plant nuclear protein, has been shown to regulate multiple biological functions in plants such as circadian rhythm, light signaling, cold tolerance, hormone signaling, and photoperiodic flowering. Recently, the role of GI in disease tolerance against different pathogens has come to light; however, a detailed mechanism to understand the role of GI in pathogen defense remains largely unexplained. Here, we report that GIGANTEA is upregulated upon infection with a virulent oomycete pathogen, Hyaloperonospora arabidopsidis (Hpa), in Arabidopsis thaliana accession Col-0. To investigate the role of GI in Arabidopsis defense, we examined the pathogen infection phenotype of gi mutant plants and found that gi-100 mutant was highly susceptible to Hpa Noco2 infection. Notably, the quantitative real-time PCR showed that PHYTOALEXIN DEFICIENT4 (PAD4) and several PAD4-regulated downstream genes were downregulated upon Noco2 infection in gi-100 mutant as compared to Col-0 plants. Furthermore, the chromatin immunoprecipitation results show that GI can directly bind to the intronic region of the PAD4 gene, which might explain the mechanism of GI function in regulating disease resistance in plants. Taken together, our results suggest that GI expression is induced upon Hpa pathogen infection and GI can regulate the expression of PAD4 to promote resistance against the oomycete pathogen Hyaloperonospora arabidopsidis in Arabidopsis thaliana.

MAP KINASE PHOSPHATASE1 promotes osmotolerance by suppressing PHYTOALEXIN DEFICIENT4-independent immunity.

Initial exposure of plants to osmotic stress caused by drought, cold, or salinity leads to acclimation, termed acquired tolerance, to subsequent severe stresses. Acquired osmotolerance induced by salt stress is widespread across Arabidopsis (Arabidopsis thaliana) accessions and is conferred by disruption of a nucleotide-binding leucine-rich repeat gene, designated ACQUIRED OSMOTOLERANCE. De-repression of this gene under osmotic stress causes detrimental autoimmunity via ENHANCED DISEASE SUSCEPTIBILITY1 and PHYTOALEXIN DEFICIENT4 (PAD4). However, the mechanism underlying acquired osmotolerance remains poorly understood. Here, we isolated an acquired osmotolerance-defective mutant (aod13) by screening 30,000 seedlings of an ion beam-mutagenized M2 population of Bu-5, an accession with acquired osmotolerance. We found that AOD13 encodes the dual-specificity phosphatase MAP KINASE PHOSPHATASE1 (MKP1), which negatively regulates MITOGEN-ACTIVATED PROTEIN KINASE3/6 (MPK3/6). Consistently, MPK3/6 activation was greater in aod13 than in the Bu-5 wild-type (WT). The aod13 mutant was sensitive to osmotic stress but tolerant to salt stress. Under osmotic stress, pathogenesis-related genes were strongly induced in aod13 but not in the Bu-5 WT. Loss of PAD4 in pad4 aod13 plants did not restore acquired osmotolerance, implying that activation of immunity independent of PAD4 renders aod13 sensitive to osmotic stress. These findings suggest that AOD13 (i.e. MKP1) promotes osmotolerance by suppressing the PAD4-independent immune response activated by MPK3/6.

A truncated CC-NB-ARC gene TaRPP13L1-3D positively regulates powdery mildew resistance in wheat via the RanGAP-WPP complex-mediated nucleocytoplasmic shuttle.

A wheat RPP13-like isoform interacting with WPP1 contributes to quantitative and/or basal resistance to powdery mildew (Blumeria graminis f. sp. tritici) by restricting the development of Bgt conidia. Plant disease resistance (R) genes confer an ability to resist infection by pathogens expressing specific avirulence genes. Recognition of Peronospora parasitica 13-like (RPP13-like) genes belong to the nucleotide-binding site and leucine-rich repeat (NBS-LRR) superfamily and play important roles in resistance to various plant diseases. Previously, we detected a TaRPP13-like gene located on chromosome 3D (TaRPP13L1-3D) in the TaSpl1 resided region, which is strongly induced by the cell death phenotype (Zhang et al. 2021). Here, we investigated the expression and functional role of TaRPP13L1-3D in wheat responding to fungal stress. TaRPP13L1-3D encoded a typical NB-ARC structure characterized by Rx-N and P-loop NTPase domains. TaRPP13L1-3D transcripts were strongly upregulated in wheat by powdery mildew (Blumeria graminis f. sp. tritici; Bgt) and stripe rust (Puccinia striiformis f. sp. tritici; Pst) infection although opposing expression patterns were observed in response to wheat-Bgt in incompatible and compatible backgrounds. Overexpression of TaRPP13L1-3D enhanced disease resistance to Bgt, accompanied by upregulation of the defense-related marker genes encoding phytoalexin-deficient4 (PAD4), thaumatin-like protein (TLP) and chitinase 8-like protein (Chi8L), while silencing of TaRPP13L1-3D disrupted the resistance to Bgt infection. Subcellular localization studies showed that TaRPP13L1-3D is located in both the plasma membrane and nucleus, while yeast-two-hybrid (Y2H) assays indicated that TaRPP13L1-3D interacts with WPP domain-containing protein 1 (TaWPP1). This indicates that TaRPP13L1-3D shuttles between the nucleus and cytoplasm membrane via a mechanism that is mediated by the RanGAP-WPP complex in nuclear pores. This insight into TaRPP13L1-3D will be useful in dissecting the mechanism of fungal resistance in wheat, and understanding the interaction between R gene expression and pathogen defense.

Glucose sensor MdHXK1 activates an immune response to the fungal pathogen Botryosphaeria dothidea in apple.

Sugars are essential regulatory molecules involved in plant growth and development and defense response. Although the relationship between sugars and disease resistance has been widely discussed, the underlying molecular mechanisms remain unexplored. Ring rot caused by Botryosphaeria dothidea (B. dothidea), which severely affects fruit quality and yield, is a destructive disease of apples (Malus domestica Borkh.). The present study found that the degree of disease resistance in apple fruit was closely related to glucose content. Therefore, the gene encoding a hexokinase, MdHXK1, was isolated from the apple cultivar 'Gala', and characterized during the defense response. Overexpression of MdHXK1 enhanced disease resistance in apple calli, leaves and fruits by increasing the expression levels of genes related to salicylate (SA) synthesis (PHYTOALEXIN DEFICIENT 4, PAD4; PHENYLALANINE AMMONIA-LYASE, PAL; and ENHANCED DISEASE SUSCEPTIBILITY 1, EDS1) and signaling (PR1; PR5; and NONEXPRESSER OF PR GENES 1, NPR1) as well as increasing the superoxide (O2- ) production rate and the hydrogen peroxide (H2 O2 ) content. Overall, the study provides new insights into the MdHXK1-mediated molecular mechanisms by which glucose signaling regulates apple ring rot resistance.

Identification of defense related gene families and their response against powdery and downy mildew infections in Vitis vinifera

BackgroundGrapevine (Vitis vinifera) productivity has been severely affected by various bacterial, viral and fungal diseases worldwide. When a plant is infected with the pathogen, various defense mechanisms are subsequently activated in plants at various molecular levels. Thus, for substantiating the disease control in an eco-friendly way, it is essential to understand the molecular mechanisms governing pathogen resistance in grapes.ResultsIn our study, we performed genome-wide identification of various defensive genes expressed during powdery mildew (PM) and downy mildew (DM) infections in grapevine. Consequently, we identified 6, 21, 2, 5, 3 and 48 genes of Enhanced Disease Susceptibility 1 (EDS1), Non-Race-specific Disease Resistance (NDR1), Phytoalexin deficient 4 (PAD4), Nonexpressor of PR Gene (NPR), Required for Mla-specified resistance (RAR) and Pathogenesis Related (PR), respectively, in the grapevine genome. The phylogenetic study revealed that V. vinifera defensive genes are evolutionarily related to Arabidopsis thaliana. Differential expression analysis resulted in identification of 2, 4, 7, 2, 4, 1 and 7 differentially expressed Nucleotide-binding leucine rich repeat receptor (NLR), EDS1, NDR1, PAD4, NPR, RAR1 and PR respectively against PM infections and 28, 2, 5, 4, 1 and 19 differentially expressed NLR, EDS1, NDR1, NPR, RAR1 and PR respectively against DM infections in V. vinifera. The co-expression study showed the occurrence of closely correlated defensive genes that were expressed during PM and DM stress conditions.ConclusionThe PM and DM responsive defensive genes found in this study can be characterized in future for impelling studies relaying fungal and oomycete resistance in plants, and the functionally validated genes would then be available for conducting in-planta transgenic gene expression studies for grapes.

ISWI chromatin remodeling factors repress PAD4-mediated plant immune responses in Arabidopsis

Chromatin remodeling proteins modulate nucleosome dynamic to affect global gene expression and other cellular processes. Their roles in the regulation of plant growth and development have been widely reported, but their roles in plant stress resistance, especially disease resistance, have not been extensively investigated. Here, we show that the Arabidopsis Immunity Switch (ISWI) chromatin-remodeling factors CHR11 and CHR17, are negative regulators of plant disease resistance. The loss of both CHR11 and CHR17 function led to upregulation of a large set of defense response genes in the absence of pathogen infection. The chr11 single mutant showed enhanced resistance against a virulent pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). Further analysis revealed that mutation of Phytoalexin Deficient4 (PAD4) reduced the upregulation of defense gene expression as well as resistance against Pst DC3000 in the chr11 chr17 double mutant. However, mutation of PAD4 does not rescue the growth defects of chr11 chr17. Together, our study revealed a function of ISWI in repressing defense response under non-pathogenic conditions and indicates distinct target genes of ISWI in regulating plant growth and plant immunity.