PR1 Expression Research Articles

Root treatment with a vitamin K3 derivative: a promising alternative to synthetic fungicides against Botrytis cinerea in tomato plants.

Botrytis cinerea, the causal agent of gray mold has a great economic impact on several important crops. This necrotrophic fungus causes disease symptoms during vegetative growth and also into postharvest stages. The current method to combat this disease is fungicide application, with high economic costs and environmentally unsustainable impacts. Moreover, there is an increasing general public health concern about these strategies of crop protection. We studied the protection of tomato plants against B. cinerea by previous root treatment with menadione sodium bisulfite (MSB), a known plant defense activator. Root treatment 48 h before inoculation with MSB 0.6 mmol L-1 reduced leaf lesion diameter by 30% and notably cell deaths, compared to control plants 72 h after inoculation. We studied the expression level of several pathogenesis-related (PR) genes from different defense transduction pathways, and found that MSB primes higher PR1 expression against B. cinerea. However, this stronger induced resistance was impaired in transgenic salicylic acid-deficient NahG line. Additionally, in the absence of pathogen challenge, MSB increased tomato plant growth by 28% after 10 days. Our data provide evidence that MSB protects tomato plants against B. cinerea by priming defense responses through the salicylic acid (SA)-dependent signaling pathway and reducing oxidative stress. This work confirms the efficacy of MSB as plant defense activator against B. cinerea and presents a novel alternative to combat gray mold in important crops.

Trichoderma-Induced Ethylene Responsive Factor MsERF105 Mediates Defense Responses in Malus sieversii.

Trichoderma can induce plant hormone signal pathways mediating plant defenses, resulting in broad-spectrum resistance to phytopathogens. Herein, Malus sieversii seedlings were treated with Trichoderma biofertilizer and/or Alternaria alternata f. sp. mali, and transcriptome analysis revealed significant differential expression. There was a high similarity between the transcriptome expression profiles of Trichoderma-induced and A. alternata-infected M. sieversii samples for genes related to jasmonic acid (JA), ethylene, and salicylic acid (SA) signaling pathways. Additionally, Trichoderma biofertilizer activated numerous disease-resistant genes (ERF, NAC, bHLH, and STK) and defense response genes (DRP, ABC, and HSP). Among transcription factors, members of the ERF family were the most differentially expressed (18 ERFs), indicating that they may be closely related to defense responses. Among ERFs, differential expression of MsERF105 was the most significant (upregulated 27.6-fold compared to controls). MsERF105 was heterologously expressed in PdPap poplar (Populus davidiana × Populus alba var. pyramidalis Louche), and following infection with A. alternata (Aal), transgenic PdPap-MsERF105s plants displayed lower malondialdehyde (downregulated 41.4%) and reactive oxygen species (ROSs) levels, and higher reductase activities, especially superoxide dismutase (SOD; upregulated 77.5% compared to PdPap-ROK2 plants). Furthermore, the lesion areas of PdPap-MsERF105s leaves were significantly smaller (0.2%) than those of PdPap-ROK2 leaves (∼26.0%), and the cell membrane integrity was superior for PdPap-MsERF105s leaves. Thus, MsERF105 enhanced the resistance of PaPap poplar to Aal, presumably because MsERF105 activates the expression of PR1 and PDF1.2. In conclusion, Trichoderma biofertilizer modulated the differential expression of numerous disease resistance genes and defense response genes in M. sieversii in response to pathogen attack, and MsERF105 played important roles in this process.

Elucidation of the molecular responses during the primary infection of wild blueberry phenotypes with Monilinia vaccinii-corymbosi under field conditions

BackgroundMonilinia blight caused by Monilinia vaccinii-corymbosi (Reade) Honey (M.vc) is a major disease of wild blueberry that can result in severe crop losses in the absence of an integrated disease management programme. The fungus causes blight in the emerging floral and vegetative buds, but the degree of susceptibility varies among the different wild blueberry phenotypes, ranging from the highly susceptible V. a. f. nigrum to the moderately susceptible V. angustifolium and the least susceptible V. myrtilloides.ResultsThe present study evaluated the defense responses of these major phenotypes during their primary infection (floral buds) with M.vc. The temporal expression profiles of PR genes (PR3 and PR4) and the flavonoid pathway structural genes (CHS, ANS, ANR, DFR and FLS) were analysed. The PR3 and PR4 gene expression profiles revealed that V. myrtilloides responded to M.vc infection by activating the expression of both PR genes. V. a. f. nigrum, on the other hand, failed to activate these genes, while V. angustifolium, exhibited an intermediate response. Our study with the flavonoid pathway genes indicated variability in activation of the genes during post-infection time points with ANS and ANR in V. myrtilloides, FLS in V. angustifolium and no response observed in V. a. f. nigrum.ConclusionsAltogether, this study highlights that the degree of phenotype susceptibility is associated with the timely activation of host defense responsive genes. Data obtained in this study provided a starting point for a better understanding of the wild blueberry- M. vaccinii-corymbosi pathosystem.

Effect of Plant Systemic Resistance Elicited by Biological and Chemical Inducers on the Colonization of the Lettuce and Basil Leaf Apoplast by Salmonella enterica.

Mitigation strategies to prevent microbial contamination of crops are lacking. We tested the hypothesis that induction of plant systemic resistance by biological (induced systemic resistance [ISR]) and chemical (systemic acquired resistance [SAR]) elicitors reduces endophytic colonization of leaves by Salmonella enterica serovars Senftenberg and Typhimurium. S. Senftenberg had greater endophytic fitness than S. Typhimurium in basil and lettuce. The apoplastic population sizes of serovars Senftenberg and Typhimurium in basil and lettuce, respectively, were significantly reduced approximately 10- to 100-fold by root treatment with microbial inducers of systemic resistance compared to H2O treatment. Rhodotorula glutinis effected the lowest population increases of S. Typhimurium in lettuce and S. Senftenberg in basil leaves, respectively 120- and 60-fold lower than those seen with the H2O treatment over 10 days postinoculation. Trichoderma harzianum and Pichia guilliermondii did not have any significant effect on S. Senftenberg in the basil apoplast. The chemical elicitors acidobenzolar-S-methyl and dl-β-amino-butyric acid inhibited S. Typhimurium multiplication in the lettuce apoplast 10- and 2-fold, respectively, compared to H2O-treated plants. All ISR and SAR inducers applied to lettuce roots in this study increased leaf expression of the defense gene PR1, as did Salmonella apoplastic colonization in H2O-treated lettuce plants. Remarkably, both acidobenzolar-S-methyl upregulation and R. glutinis upregulation of PR1 were repressed by the presence of Salmonella in the leaves. However, enhanced PR1 expression was sustained longer and at greater levels upon elicitor treatment than by Salmonella induction alone. These results serve as a proof of concept that priming of plant immunity may provide an intrinsic hurdle against the endophytic establishment of enteric pathogens in leafy vegetables. IMPORTANCE Fruit and vegetables consumed raw have become an important vehicle of foodborne illness despite a continuous effort to improve their microbial safety. Salmonella enterica has caused numerous recalls and outbreaks of infection associated with contaminated leafy vegetables. Evidence is increasing that enteric pathogens can reach the leaf apoplast, where they confront plant innate immunity. Plants may be triggered for induction of their defense signaling pathways by exposure to chemical or microbial elicitors. This priming for recognition of microbes by plant defense pathways has been used to inhibit plant pathogens and limit disease. Given that current mitigation strategies are insufficient in preventing microbial contamination of produce and associated outbreaks, we investigated the effect of plant-induced resistance on S. enterica colonization of the lettuce and basil leaf apoplast in order to gain a proof of concept for the use of such an intrinsic approach to inhibit human pathogens in leafy vegetables.

The Fusarium graminearum FGSG_03624 Xylanase Enhances Plant Immunity and Increases Resistance against Bacterial and Fungal Pathogens.

Fungal enzymes degrading the plant cell wall, such as xylanases, can activate plant immune responses. The Fusarium graminearum FGSG_03624 xylanase, previously shown to elicit necrosis and hydrogen peroxide accumulation in wheat, was investigated for its ability to induce disease resistance. To this aim, we transiently and constitutively expressed an enzymatically inactive form of FGSG_03624 in tobacco and Arabidopsis, respectively. The plants were challenged with Pseudomonas syringae pv. tabaci or pv. maculicola and Botrytis cinerea. Symptom reduction by the bacterium was evident, while no reduction was observed after B. cinerea inoculation. Compared to the control, the presence of the xylanase gene in transgenic Arabidopsis plants did not alter the basal expression of a set of defense-related genes, and, after the P. syringae inoculation, a prolonged PR1 expression was detected. F. graminearum inoculation experiments of durum wheat spikes exogenously treated with the FGSG_03624 xylanase highlighted a reduction of symptoms in the early phases of infection and a lower fungal biomass accumulation than in the control. Besides, callose deposition was detected in infected spikes previously treated with the xylanase and not in infected control plants. In conclusion, our results highlight the ability of FGSG_03624 to enhance plant immunity, thus decreasing disease severity.

Pseudomonas Inoculation Stimulates Endophytic Azospira Population and Induces Systemic Resistance to Bacterial Wilt.

Bacterial communities in the rhizosphere play an important role in sustaining plant growth and the health of diverse soils. Recent studies have demonstrated that microbial keystone taxa in the rhizosphere microbial community are extremely critical for the suppression of diseases. However, the mechanisms involved in disease suppression by keystone species remain unclear. The present study assessed the effects of three Pseudomonas strains, which were identified as keystone species in our previous study, on the growth performance and root-associated bacterial community of tobacco plants. A high relative abundance of Ralstonia was found in the non-inoculated group, while a large Azospira population was observed in all groups inoculated with the three Pseudomonas strains. Correspondingly, the activities of the defense-related enzymes and the expression levels of the defense signaling marker genes of the plant were increased after inoculation with the Pseudomonas strains. Moreover, the correlation analyses showed that the relative abundance of Azospira, the activity of superoxide dismutase, catalase, and polyphenol oxidase, and the expression of H1N1, ACC Oxidase, and PR1 a/c had a significantly negative (p<0.05) relationship with the abundance of Ralstonia. This further revealed that the keystone species, such as Pseudomonas spp., can suppress bacterial wilt disease by enhancing the systemic resistance of tobacco plants.

Multifunctional efficacy of the nodule endophyte Pseudomonas fragi in stimulating tomato immune response against Meloidogyne incognita

Root-knot nematodes severely affect the growth and productivity of agricultural plants in the world. Conversely, endophytic bacteria can be used to prevent plant diseases and increase crop yield. The nodule endophytic bacterium Pseudomonas fragi (Eichholz) Gruber has been reported to promote crop growth, but its role in nematode control is unclear. In this study, the biocontrol effect of P. fragi Sneb1990 against Meloidogyne incognita (Kofoid & White) Chitwood was assessed in tomato (Solanum lycopersicum L.). In vitro, the fermentation broth of P. fragi Sneb1990 provoked 76.8% juvenile mortality after 48 h and 71.9% egg mortality after 7 d. P. fragi Sneb1990 promoted plant growth and alleviated M. incognita disease symptoms in pot and field experiments. The split-root assay revealed that P. fragi Sneb1990 stimulated local and systemic immunity to suppress the penetration and development of M. incognita and that it enhanced the expression of PR1, PR2, PR3, PR5, and PDF1.2, which participate in the jasmonic acid and salicylic acid pathways. Furthermore, the flagellin polypeptide flg22 of P. fragi Sneb1990 triggered tomato immunity against M. incognita, stimulated reactive oxygen species production, and upregulated the expression of salicylic acid resistance-related genes. This is the first report of the suppression of M. incognita infection by the nodule endophyte P. fragi. In addition, the flagellin functional domain of P. fragi Sneb1990 triggered immune responses against M. incognita in tomato. Overall, our study highlights the potential of P. fragi Sneb1990 as a biocontrol agent against M. incognita.

Monochromic Radiations Provided by Light Emitted Diode (LED) Modulate Infection and Defense Response to Fire Blight in Pear Trees.

Pathogenesis-related (PR) proteins are part of the systemic signaling network that perceives pathogens and activates defenses in the plant. Eukaryotic and bacterial species have a 24-h ‘body clock’ known as the circadian rhythm. This rhythm regulates an organism’s life, modulating the activity of the phytochromes (phys) and cryptochromes (crys) and the accumulation of the corresponding mRNAs, which results in the synchronization of the internal clock and works as zeitgeber molecules. Salicylic acid accumulation is also under light control and upregulates the PR genes expression, increasing plants’ resistance to pathogens. Erwinia amylovora causes fire blight disease in pear trees. In this work, four bacterial transcripts (erw1-4), expressed in asymptomatic E. amylovora-infected pear plantlets, were isolated. The research aimed to understand how the circadian clock, light quality, and related photoreceptors regulate PR and erw genes expression using transgenic pear lines overexpressing PHYB and CRY1 as a model system. Plantlets were exposed to different circadian conditions, and continuous monochromic radiations (Blue, Red, and Far-Red) were provided by light-emitting diodes (LED). Results showed a circadian oscillation of PR10 gene expression, while PR1 was expressed without clear evidence of circadian regulation. Bacterial growth was regulated by monochromatic light: the growth of bacteria exposed to Far-Red did not differ from that detected in darkness; instead, it was mildly stimulated under Red, while it was significantly inhibited under Blue. In this regulatory framework, the active form of phytochrome enhances the expression of PR1 five to 15 fold. An ultradian rhythm was observed fitting the zeitgeber role played by CRY1. These results also highlight a regulating role of photoreceptors on the expression of PRs genes in non-infected and infected plantlets, which influenced the expression of erw genes. Data are discussed concerning the regulatory role of photoreceptors during photoperiod and pathogen attacks.

Convergent Epigenetic Mechanisms Avoid Constitutive Expression of Immune Receptor Gene Subsets.

The gene pool encoding PRR and NLR immune receptors determines the ability of a plant to resist microbial infections. Basal expression of these genes is prevented by diverse mechanisms since their hyperactivity can be harmful. To approach the study of epigenetic control of PRR/NLR genes we here analyzed their expression in mutants carrying abnormal repressive 5-methyl cytosine (5-mC) and histone 3 lysine 9 dimethylation (H3K9me2) marks, due to lack of MET1, CMT3, MOM1, SUVH4/5/6, or DDM1. At optimal growth conditions, none of the mutants showed basal expression of the defense gene marker PR1, but all of them had greater resistance to Pseudomonas syringae pv. tomato than wild type plants, suggesting they are primed to stimulate immune cascades. Consistently, analysis of available transcriptomes indicated that all mutants showed activation of particular PRR/NLR genes under some growth conditions. Under low defense activation, 37 PRR/NLR genes were expressed in these plants, but 29 of them were exclusively activated in specific mutants, indicating that MET1, CMT3, MOM1, SUVH4/5/6, and DDM1 mediate basal repression of different subsets of genes. Some epigenetic marks present at promoters, but not gene bodies, could explain the activation of these genes in the mutants. As expected, suvh4/5/6 and ddm1 activated genes carrying 5-mC and H3K9me2 marks in wild type plants. Surprisingly, all mutants expressed genes harboring promoter H2A.Z/H3K27me3 marks likely affected by the chromatin remodeler PIE1 and the histone demethylase REF6, respectively. Therefore, MET1, CMT3, MOM1, SUVH4/5/6, and DDM1, together with REF6, seemingly contribute to the establishment of chromatin states that prevent constitutive PRR/NLR gene activation, but facilitate their priming by modulating epigenetic marks at their promoters.

The Capsicum baccatum-Specific Truncated NLR Protein CbCN Enhances the Innate Immunity against Colletotrichum acutatum.

Chili pepper (Capsicum annuum) is an important fruit and spice used globally, but its yield is seriously threatened by anthracnose. Capsicum baccatum is particularly valuable as it carries advantageous disease resistance genes. However, most of the genes remain to be identified. In this study, we identified the C. baccatum-specific gene CbCN, which encodes a truncated nucleotide-binding and leucine-rich repeat protein in the anthracnose resistant chili pepper variety PBC80. The transcription of CbCN was greater in PBC80 than it was in the susceptible variety An-S after Colletotrichum acutatum inoculation. In order to investigate the biological function of CbCN, we generated transgenic tobacco lines constitutively expressing CbCN. Notably, CbCN-overexpressing transgenic plants exhibited enhanced resistance to C. acutatum compared to wild-type plants. Moreover, the expression of pathogenesis-related (PR) genes was remarkably increased in a CbCN-overexpressing tobacco plants. In order to confirm these results in chili pepper, we silenced the CbCN gene using the virus-induced gene silencing system. The anthracnose resistance and expressions of PR1, PR2, and NPR1 were significantly reduced in CbCN-silenced chili peppers after C. acutatum inoculations. These results indicate that CbCN enhances the innate immunity against anthracnose caused by C. acutatum by regulating defense response genes.

AtWRKY1 negatively regulates the response of Arabidopsis thaliana to Pst. DC3000

WRKY transcription factors (TFs) play a major role in resistance to plant diseases, but the role of AtWRKY1 in response to Pst. DC3000 is not clear. In this study, we found that AtWRKY1 negatively affected the response of Arabidopsis to Pst. DC3000. During Pst. DC3000 infection, the transcription of AtWRKY1 was suppressed. The wrky1 mutants displayed enhanced resistance to Pst. DC3000. In contrast, the overexpression of AtWRKY1 reduced the resistance. The relative RNA levels of defense related PR genes were increased in the loss-of-function mutants, whereas their expressions were decreased in the AtWRKY1-overexpressing plants. Further research revealed that salicylic acid (SA) can repress the expression of AtWRKY1, and overexpression of AtWRKY1 weakened the SA-mediated defense response. In addition, the AtWRKY1 protein can bind to the PR1 promoter in vivo and in yeast cells directly, thereby inhibiting the transcription of PR1. AtWRKY1 indirectly represses the expression of PR2 and PR5. Our results indicated that the AtWRKY1 gene negatively regulates the plant defense responses to Pst. DC3000 through SA signaling pathways.

Electrical Stimulation Enhances Plant Defense Response in Grapevine through Salicylic Acid-Dependent Defense Pathway.

Concern over environmental pollution generated by chemical fungicides has led to the introduction of alternative pest management strategies to chemical fungicide application. One of those strategies is the induction of plant defense response by an abiotic elicitor. In the present study, field-grown grapevines were subjected to electrical stimulation using a solar panel from two weeks before flowering to harvest in the 2016 and 2020 growing seasons. In both years, electrical stimulation decreased the incidence of gray mold and/or ripe rot on bunches and downy mildew on leaves of the field-grown grapevine. Transcription of a gene encoding β-1,3-glucanase but not class IV chitinase in leaves of potted grapevine seedlings was upregulated 20 days after electrical stimulation, suggesting that electrical stimulation acts as an abiotic elicitor of plant defense response to fungal diseases. The gene expression of PR1 but not PDF1.2 was upregulated in Arabidopsis plants subjected to electrical stimulation. On the other hand, PR1 gene expression was not induced in salicylic acid (SA)-insensitive Arabidopsis mutant npr1-5 subjected to electrical stimulation. Taken together, electrical stimulation is responsible for plant defense response through the SA-dependent defense pathway. These findings would help us develop a novel and innovative practical technique that uses electrical stimulation in integrated pest management.

Cathepsin G Is Expressed By B Cell Acute Lymphoblastic Leukemia and Is an Effective Immunotherapeutic Target

The azurophil granule proteases neutrophil elastase (NE) and proteinase 3 (P3) have been established as effective immunotherapeutic targets in hematological malignancies. Expression of PR1, an HLA-A2 restricted nonameric peptide derived from NE and P3, has been demonstrated in acute myeloid leukemia (AML). We have successfully targeted PR1 in AML patients using PR1-peptide vaccine, anti-PR1/HLA-A2 antibody and PR1-specific cytotoxic T-lymphocytes (PR1-CTL). We have also identified the uptake and cross-presentation of NE and P3 by breast cancer, lung cancer and melanoma, which underlines their potential as immunotherapeutic targets in non-myeloid malignancies.Cathepsin G is also an azurophil granule serine protease that is highly expressed by AML blasts and leukemic stem cells. Our previous studies showed a high level of efficacy in targeting AML blasts that express CG1 (FLLPTGAEA) using CG1-specific CTL (CG1-CTL), while sparing normal hematopoietic cells. However, two previous reports (Fujiwara et al., Clinical Cancer Research, 2005 and Gorodkiewicz et al., Analytical Biochemistry, 2012) and our published data (Alatrash G, Leukemia, 2017) have reported CG expression in lymphoid leukemia. We therefore hypothesized that CG is an effective immunotherapeutic target in lymphoid malignancies, specifically acute lymphoid leukemia (ALL).We first analyzed CG expression in ALL patient samples. Using reverse phase protein array (RPPA), we analyzed CG expression in ALL blasts from 345 adult ALL patient samples. We detected CG in 345 patient samples at levels comparable to G-CSF stimulated myeloid progenitors. The differential expression of CG in sorted ALL blasts ranged from -1.8030 to 5.4390, with a median value of 0.3506. In estimating CG levels, we found that more than 15% of our ALL samples expressed Log2 concentrations of CG between 0.3 and 0.4 (Figure 1). This was comparable to what we have previously reported in AML. We then wanted to determine whether the ALL-associated CG was due to uptake or endogenous expression. We performed PCR and western blotting and confirmed CG expression in 4 of 9 and 9 of 9 patient ALL samples, respectively (Figure 2). To assess whether CG is also taken up by ALL, we examined CG uptake by the B-ALL cell lines SB, RS4, Nalm6 and HMY (B-ALL). ALL cell lines were incubated with CG protein at increasing doses and durations. Intracellular staining of CG followed by flow cytometry analysis was used to determine uptake of CG by ALL cell lines. Our data show that all of the ALL cell lines take up CG; uptake correlated directly with increasing doses and time of incubation up to 24 hours.Having characterized the expression of CG by ALL, we sought to determine its potential as an immunotherapeutic target, as we have previously shown for AML. In these experiments, CG1-specific CTLs were generated using standard dendritic cells methodology, interleukin-7 (IL-7), IL-2 and CG1-peptide pulsing. Expanded CG1-CTL were confirmed by staining cells with CG1 tetramer followed by flow cytometry analysis. We employed standard calcein-AM cytotoxicity assays to determine specific lysis. In these experiments, patient ALL sample UPN8 was pulsed with calcein-AM and then co-cultured with CG1-CTL for 4 hours. Our results indicated that UPN8 was effectively lysed by CG1-CTL, reaching a maximum specific lysis of ~30% at the highest effector:target ratio (Figure 3).Overall, our analysis confirms CG endogenous expression and uptake by ALL. Furthermore, we conclude that CG is an effective target for immunotherapy in B-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Roles of Endogenous Melatonin in Resistance to Botrytis cinerea Infection in an Arabidopsis Model.

Melatonin is an important bioactive molecule in plants. Two synthetases, N-acetylserotonin methyltransferase (ASMT) and serotonin N-acetyltransferase (SNAT) are involved in the final two steps of melatonin synthesis. Melatonin participates in responses to a variety of biotic and abiotic stresses in plants, but few studies have addressed the roles of endogenous melatonin in pathogen resistance. We investigated the role of endogenous melatonin in resistance to Botrytis cinerea infection in an Arabidopsis thaliana model system. Plant lines that overexpressed ASMT or SNAT through genetic manipulation showed upregulated expression of resistance genes PR1 and PR5, transcription factor gene WRKY33, and jasmonic acid (JA) defense pathway marker gene PDF1.2, and downregulated transcription factor gene MYC2 in JA signaling pathway. Higher melatonin content also enhanced the activity of antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD), increased JA content, reduced plant disease symptoms, and reduced lesion size in leaves. These findings indicate that endogenous melatonin enhances plant resistance to B. cinerea infection. In contrast, ASMT and SNAT gene silencing lines showed opposite results and were more susceptible to B. cinerea. Thus, it can be demonstrated that melatonin functions as an effective regulator of plant stress resistance at the genetic level. A schematic model is presented for its role in resistance to B. cinerea infection. Our findings also helped to elucidate the associated signal transduction pathways and interactions between melatonin and other plant hormones.

Sulfur Deprivation Modulates Salicylic Acid Responses via Nonexpressor of Pathogenesis-Related Gene 1 in Arabidopsis thaliana.

Mineral nutrients are essential for plant growth and reproduction, yet only a few studies connect the nutritional status to plant innate immunity. The backbone of plant defense response is mainly controlled by two major hormones: salicylic acid (SA) and jasmonic acid (JA). This study investigated changes in the macronutrient concentration (deficiency/excess of nitrogen, phosphorus, potassium, magnesium, and sulfur) on the expression of PR1, a well-characterized marker in the SA-pathway, and PDF1.2 and LOX2 for the JA-pathway, analyzing plants carrying the promoter of each gene fused to GUS as a reporter. After histochemical GUS assays, we determined that PR1 gene was strongly activated in response to sulfur (S) deficiency. Using RT-PCR, we observed that the induction of PR1 depended on the function of Non-expressor of Pathogenesis-Related gene 1 (NPR1) and SA accumulation, as PR1 was not expressed in npr1-1 mutant and NahG plants under S-deprived conditions. Plants treated with different S-concentrations showed that total S-deprivation was required to induce SA-mediated defense responses. Additionally, bioassays revealed that S-deprived plants, induced resistance to the hemibiotrophic pathogen Pseudomonas syringae pv. DC3000 and increase susceptibility to the necrotrophic Botrytis cinerea. In conclusion, we observed a relationship between S and SA/JA-dependent defense mechanisms in Arabidopsis.

BRASSINOSTEROID‐SIGNALLING KINASES 7 and 8 associate with the FLS2 immune receptor and are required for flg22‐induced PTI responses

Pattern‐triggered immunity (PTI) is typically initiated in plants by recognition of pathogen‐ or damage‐associated molecular patterns (PAMP/DAMPs) by cell surface‐localized pattern recognition receptors (PRRs). Here, we investigated the role in PTI of Arabidopsis thaliana brassinosteroid‐signalling kinases 7 and 8 (BSK7 and BSK8), which are members of the receptor‐like cytoplasmic kinase subfamily XII. BSK7 and BSK8 localized to the plant cell periphery and interacted in yeast and in planta with FLS2, but not with other PRRs. Consistent with a role in FLS2 signalling, bsk7 and bsk8 single and bsk7,8 double mutant plants were impaired in several immune responses induced by flg22, but not by other PAMP/DAMPs. These included resistance to Pseudomonas syringae and Botrytis cinerea, reactive oxygen species accumulation, callose deposition at the cell wall, and expression of the defence‐related gene PR1, but not activation of MAP kinases and expression of the FRK1 and WRKY29 genes. bsk7, bsk8, and bsk7,8 plants also displayed enhanced susceptibility to P. syringae and B. cinerea. Finally, BSK7 and BSK8 variants mutated in their myristoylation site or in the ATP‐binding site failed to complement defective phenotypes of the corresponding mutants, suggesting that localization to the cell periphery and kinase activity are critical for BSK7 and BSK8 functions. Together, these findings demonstrate that BSK7 and BSK8 play a role in PTI initiated by recognition of flg22 by interacting with the FLS2 immune receptor.

Fine mapping and candidate gene analysis of leaf tip premature senescence and Dwarf Mutant dls-1 in Rice

Premature leaf senescence negatively affects rice yield and quality. Identifying premature senescence mutants and examining their gene functions are important for the genetic improvement of crops. In this study, a leaf senescence mutant dls-1 was obtained from japonica Yundao by ethyl methane sulfonate mutagenesis, which showed the phenotypes of premature leaf senescence, reduced plant height and decreased yield-associated traits including panicle length, seed setting rate, and 1000-grain weight. Biochemical analysis revealed the accumulation of reactive oxygen species in dls-1 leading to increased apoptosis rates, stomata length extension, higher water loss, and a decline in leaf silicification. Transcriptomic data showed that pathogen resistance-related genes were down-regulated in dls-1, causing increased sensitivity to bacterial-induced blight. Genetic analysis indicated that the mutant trait of dls-1 was controlled by a single recessive nuclear gene, and the gene was fine-mapped to a 159 kb region on rice chromosome 1 using map-based cloning strategy. A total of 23 open reading frames were found in the interval, and no similar phenotypic genes have been reported. However, all candidate coding genes and promoter regions were sequenced, and no mutation sites were found. RNA-seq analysis and qRT-PCR showed that the candidate gene LOC_Os01g71670, encodes a rice endo-1,3-β-glucanase, showed significantly different expression levels in dls-1 compared to the wild-type. LOC_Os01g71670 is highly homologous with GII in barley, and Gns4 and glu1 in rice, which participates in the hormone response pathway affecting plant growth and development, and also involve in the regulation of PR2 expression. Taken together with the phenotypes of dls-1, we conclude that LOC_Os01g71670 may be the candidate gene, causing the premature senescence of the leaf tip and loss of pathogen resistance.

Molecular and biochemical insight into biochar and Bacillus subtilis induced defense in tomatoes against Alternaria solani

This study was aimed to elucidate the biochar induced resistance pathways in association with beneficial plant growth promoting rhizobacteria (PGPR) in the tomato (Solanum lycopersicum) – Alternaria solani pathosystem. To examine the impact of biochar on plant defense mechanisms at the molecular level, tomato plants were grown in two different biochars (wood and green waste biochar; 3 and 6% application level of each) both with and without Bacillus subtilis, (PGPR). Although, the reduction in disease index and disease incidence was significant in wood biochar amended soil but the resistant plant response against early blight was only recorded in the 6% green waste biochar amended treatment. Biochar application has induced the expression of defense associated genes including jasmonic acid related PI2, TomloxD, salicylic acid related PR1a, PR2 and ethylene responsive Pti4. Green waste biochar (6%) was more efficient in eliciting the expression level of TomloxD, PI2, PR1a and PR2 in tomatoes challenged by A. solani, while PGPR inoculation has enhanced the PR2 expression levels in comparison to tomatoes grown in wood biochar amended soil substrate. Moreover, phenolics, catalase and peroxidase production levels of plants grown in green waste biochar alone as well as in the presence of B. subtilis, contributed significantly in moderating infection induced damages.In conclusion, tomatoes response to invading A. solani was dependent on the type of biochar, while protection against early blight was better at higher application rate of biochar i.e. 6%. Different biochar types also differ with their ability to elicit the defense gene expression/signaling pathways. Our study revealed that biochar and PGPR induced protection against early blight of tomatoes involves both induced systemic resistance (ISR) and systemic acquired resistance (SAR) mechanisms.

Root Endophytism by Pochonia chlamydosporia Affects Defense-Gene Expression in Leaves of Monocot and Dicot Hosts under Multiple Biotic Interactions.

A study was carried out on the effect of the root endophytic fungus Pochonia chlamydosporia on plant systemic signal of defense related genes during fungal or nematode parasitism. Different biotic stress factors were examined, inoculating roots of dicot and monocot hosts with the endophyte, and measuring the expression of defense genes in leaves. A first greenhouse assay was carried out on expression of PAL, PIN II, PR1 and LOX D in leaves of tomato cv Tondino inoculated with Phytophthora infestans (CBS 120920), inoculating or not the roots of infected plants with P. chlamydosporia DSM 26985. In a second assay, plants of banana (Musa acuminata cv Grand Naine) were artificially infected with Fusarium oxysporum f. sp. cubense Tropical race 4 (TR4) and inoculated or not with DSM 26985. In a further experiment, banana plants were inoculated or not with P. chlamydosporia plus juveniles of the root knot nematode (RKN) Meloidogyne incognita. A similar assay was also carried out in vitro with adults and juveniles of the lesion nematode Pratylenchus goodeyi. Differential expression of the defense genes examined was observed for all plant-stress associations, indicative of early, upward systemic signals induced by the endophyte. Changes in expression profiles included a 5-fold down-regulation of PIN II at 2 dai in leaves of tomato plants treated with P. infestans and/or P. chlamydosporia, and the up-regulation of PAL by the endophyte alone, at 2 and 7 dai. In the TR4 assay, PR1 was significantly up-regulated at 7 dai in banana leaves, but only in the P. chlamydosporia treated plants. At 10 dai, PIN II expression was significantly higher in leaves of plants inoculated only with TR4. The banana-RKN assay showed a PR1 expression significantly higher than controls at 4 and 7 dai in plants inoculated with P. chlamydosporia alone, and a down-regulation at 4 dai in leaves of plants also inoculated with RKN, with a PR1 differential up-regulation at 10 dai. Pratylenchus goodeyi down-regulated PIN at 21 dai, with or without the endophyte, as well as PAL but only in presence of P. chlamydosporia. When inoculated alone, the endophyte up-regulated PR1 and LOX. The gene expression patterns observed in leaves suggest specific and time-dependent relationships linking host plants and P. chlamydosporia in presence of biotic stress factors, functional to a systemic, although complex, activation of defense genes.

Opposing functions of the plant TOPLESS gene family during SNC1-mediated autoimmunity.

Regulation of the plant immune system is important for controlling the specificity and amplitude of responses to pathogens and in preventing growth-inhibiting autoimmunity that leads to reductions in plant fitness. In previous work, we reported that SRFR1, a negative regulator of effector-triggered immunity, interacts with SNC1 and EDS1. When SRFR1 is non-functional in the Arabidopsis accession Col-0, SNC1 levels increase, causing a cascade of events that lead to autoimmunity phenotypes. Previous work showed that some members of the transcriptional co-repressor family TOPLESS interact with SNC1 to repress negative regulators of immunity. Therefore, to explore potential connections between SRFR1 and TOPLESS family members, we took a genetic approach that examined the effect of each TOPLESS member in the srfr1 mutant background. The data indicated that an additive genetic interaction exists between SRFR1 and two members of the TOPLESS family, TPR2 and TPR3, as demonstrated by increased stunting and elevated PR2 expression in srfr1 tpr2 and srfr1 tpr2 tpr3 mutants. Furthermore, the tpr2 mutation intensifies autoimmunity in the auto-active snc1-1 mutant, indicating a novel role of these TOPLESS family members in negatively regulating SNC1-dependent phenotypes. This negative regulation can also be reversed by overexpressing TPR2 in the srfr1 tpr2 background. Similar to TPR1 that positively regulates snc1-1 phenotypes by interacting with SNC1, we show here that TPR2 directly binds the N-terminal domain of SNC1. In addition, TPR2 interacts with TPR1 in vivo, suggesting that the opposite functions of TPR2 and TPR1 are based on titration of SNC1-TPR1 complexes by TPR2 or altered functions of a SNC1-TPR1-TPR2 complex. Thus, this work uncovers diverse functions of individual members of the TOPLESS family in Arabidopsis and provides evidence for the additive effect of transcriptional and post-transcriptional regulation of SNC1.