PR1 Expression Research Articles

Editorial: Control of Plant Pathogens by Biogenic Elicitors and Possible Mechanisms of Action.

Editorial: Control of Plant Pathogens by Biogenic Elicitors and Possible Mechanisms of Action.

Arabidopsis thaliana: A Model Host Plant to Study Plant-Pathogen Interaction Using Rice False Smut Isolates of Ustilaginoidea virens.

Rice false smut fungus which is a biotrophic fungal pathogen causes an important rice disease and brings a severe damage where rice is cultivated. We established a new fungal-plant pathosystem where Ustilaginoidea virens was able to interact compatibly with the model plant Arabidopsis thaliana. Disease symptoms were apparent on the leaves of the plants after 6 days of post inoculation in the form of chlorosis. Cytological studies showed that U. virens caused a heavy infestation inside the cells of the chlorotic tissues. Development and colonization of aerial mycelia in association with floral organ, particularly on anther and stigma of the flowers after 3 weeks of post inoculation was evident which finally caused infection on the developing seeds and pod tissues. The fungus adopts a uniquely biotrophic infection strategy in roots and spreads without causing a loss of host cell viability. We have also demonstrated that U. virens isolates infect Arabidopsis and the plant subsequently activates different defense response mechanisms which are witnessed by the expression of pathogenesis-related genes, PR-1, PR-2, PR-5, PDF1.1, and PDF1.2. The established A. thaliana–U. virens pathosystem will now permit various follow-up molecular genetics and gene expression experiments to be performed to identify the defense signals and responses that restrict fungal hyphae colonization in planta and also provide initial evidence for tissue-adapted fungal infection strategies.

Are Bacterial Volatile Compounds Poisonous Odors to a Fungal Pathogen Botrytis cinerea, Alarm Signals to Arabidopsis Seedlings for Eliciting Induced Resistance, or Both?

Biological control (biocontrol) agents act on plants via numerous mechanisms, and can be used to protect plants from pathogens. Biocontrol agents can act directly as pathogen antagonists or competitors or indirectly to promote plant induced systemic resistance (ISR). Whether a biocontrol agent acts directly or indirectly depends on the specific strain and the pathosystem type. We reported previously that bacterial volatile organic compounds (VOCs) are determinants for eliciting plant ISR. Emerging data suggest that bacterial VOCs also can directly inhibit fungal and plant growth. The aim of the current study was to differentiate direct and indirect mechanisms of bacterial VOC effects against Botrytis cinerea infection of Arabidopsis. Volatile emissions from Bacillus subtilis GB03 successfully protected Arabidopsis seedlings against B. cinerea. First, we investigated the direct effects of bacterial VOCs on symptom development and different phenological stages of B. cinerea including spore germination, mycelial attachment to the leaf surface, mycelial growth, and sporulation in vitro and in planta. Volatile emissions inhibited hyphal growth in a dose-dependent manner in vitro, and interfered with fungal attachment on the hydrophobic leaf surface. Second, the optimized bacterial concentration that did not directly inhibit fungal growth successfully protected Arabidopsis from fungal infection, which indicates that bacterial VOC-elicited plant ISR has a more important role in biocontrol than direct inhibition of fungal growth on Arabidopsis. We performed qRT-PCR to investigate the priming of the defense-related genes PR1, PDF1.2, and ChiB at 0, 12, 24, and 36 h post-infection and 14 days after the start of plant exposure to bacterial VOCs. The results indicate that bacterial VOCs potentiate expression of PR1 and PDF1.2 but not ChiB, which stimulates SA- and JA-dependent signaling pathways in plant ISR and protects plants against pathogen colonization. This study provides new evidence for bacterial VOC-elicited plant ISR that protects Arabidopsis plants from infection by the necrotrophic fungus B. cinerea. Our work reveals that bacterial VOCs primarily act via an indirect mechanism to elicit plant ISR, and have a major role in biocontrol against fungal pathogens.

Identification of a Novel Small Cysteine-Rich Protein in the Fraction from the Biocontrol Fusarium oxysporum Strain CS-20 that Mitigates Fusarium Wilt Symptoms and Triggers Defense Responses in Tomato.

The biocontrol effect of the non-pathogenic Fusarium oxysporum strain CS-20 against the tomato wilt pathogen F. oxysporum f. sp. lycopersici (FOL) has been previously reported to be primarily plant-mediated. This study shows that CS-20 produces proteins, which elicit defense responses in tomato plants. Three protein-containing fractions were isolated from CS-20 biomass using size exclusion chromatography. Exposure of seedling roots to one of these fractions prior to inoculation with pathogenic FOL strains significantly reduced wilt severity. This fraction initiated an ion exchange response in cultured tomato cells resulting in a reversible alteration of extracellular pH; increased tomato chitinase activity, and induced systemic resistance by enhancing PR-1 expression in tomato leaves. Two other protein fractions were inactive in seedling protection. The main polypeptide (designated CS20EP), which was specifically present in the defense-inducing fraction and was not detected in inactive protein fractions, was identified. The nucleotide sequence encoding this protein was determined, and its complete amino acid sequence was deduced from direct Edman degradation (25 N-terminal amino acid residues) and DNA sequencing. The CS20EP was found to be a small basic cysteine-rich protein with a pI of 9.87 and 23.43% of hydrophobic amino acid residues. BLAST search in the NCBI database showed that the protein is new; however, it displays 48% sequence similarity with a hypothetical protein FGSG_10784 from F. graminearum strain PH-1. The contribution of CS20EP to elicitation of tomato defense responses resulting in wilt mitigating is discussed.

Multigene MRD Assessment Improves AML Relapse Risk Stratification in Autologous Hematopoietic Cell Transplantation

▪While commonly used in other hematological malignancies, high dose chemotherapy followed by autologous hematopoietic cell transplantation (auto-HCT) has not been widely adopted in acute myeloid leukemia (AML) due to concerns regarding high post-transplant relapse rates. These relapses may be due, in part, to autograft contamination with AML. High sensitivity methods to detect residual AML have demonstrated the ability to correctly identify patients in morphological complete remission (CR) at risk of relapse. We sought to determine if testing of the graft prior to transplantation could predict post auto-HCT relapse. Prior studies investigating the utility of measurable residual disease (MRD) in auto-HCT have been limited by small patient cohorts and/or by the use of a single biomarker to detect AML, a heterogeneous cancer. We report here the largest retrospective study to date of adult auto-HCT AML patients tested for measurable residual disease (MRD) by both molecular methods (RQ-PCR) and multi-parameter flow cytometry (MPFC).Seventy-two patients transplanted between 2004-2013 at a single academic medical center (UCSF) were eligible for this study based on availability of cryopreserved GCSF mobilized autologous peripheral blood progenitor cell (PBPCs) specimens. All samples were collected on IRB approved research protocols.The median age at time of transplant was 48 (24-69), 54% were female, cytogenetics were known in 94% (of which 24% were favorable, 69% intermediate, 7% poor), 99% were in CR at the time of HCT (64/72 in CR1, 6 in CR2, 1 in CR3). Following auto-HCT 1 year RFS was 50% (1 year relapse rate 43%) and 2 year RFS was 40% (2 year relapse rate 47%).Wilms tumor 1 (WT1) is expressed in up to 90% of AML, but sufficiently over-expressed in peripheral blood to have utility as a sensitive marker of MRD in less than 50% of cases. RQ-PCR detectable WT1 above the previously established European LeukemiaNet threshold was found in 9 patients, of whom 6 relapsed in the first year after auto-HCT (19% sensitivity, 93% specificity, PPV: 67%, NPV: 60%).We have previously reported that multi-gene testing can augment WT1 based MRD detection in AML. We were unable however to use one component of our previously reported AML MRD panel, PR3, due to increased baseline expression in these GCSF stimulated PBPC products. Increased PR3 expression following GCSF administration, correlating with neutrophilia, was also observed in an independent cohort of healthy donors.Testing for PRAME, MSLN, CCNA1, t(8,21), Inv16, t(15:17) and NPM1 mutations A, B and D, as a supplement for WT1, in pre-HCT PBPCs resulted in substantially improved ability to predict post auto-HCT relapse (52% sensitivity, 80% specificity, PPV: 67%, NPV: 69%). Addition of these extra genes allowed for correct identification of 10 additional MRD+ patients who relapsed within 1yr after transplant, and added 5 false positive patients (1 of whom suffered early non-relapse mortality, and another who did relapse but more than 1 year after HCT).Finally, in expert hands, flow cytometry can identify residual AML with high sensitivity. Forty PBPC samples from the above cohort were also assessed for MRD using MPFC. CD34 positive cells comprised 0.05-12.5% of autograft specimen mononuclear cells. Due to immunophenotypic changes likely attributable to GCSF mobilization, and without leukemia associated immunophenotypes from diagnosis available, MPFC was unable to identify MRD in any of 40 patients tested.In summary, no single MRD test on autografts could completely predict post-HCT AML relapse. Auto-HCT presents unique challenges for AML MRD testing due to masking effects of GCSF on MPFC and RQ-PCR gene expression signatures. Additionally, detection of any AML autograft contamination must be extremely sensitive to be useful for predicting relapse given the absence of any potentially protective graft versus leukemia effect following an auto-HCT. Here we show combinations of molecular MRD assays can overcome some, but not all, of these limitations (Figure). [Display omitted] DisclosuresRadich:Incyte: Consultancy; Ariad: Consultancy; Novartis: Consultancy; Novartis: Other: Lab Contract. Andreadis:Cellerant: Consultancy; Novartis: Consultancy; McGraw Hill: Other: Publishers; Pharmacyclics: Honoraria. Damon:Sunesis: Research Funding; McGraw Hill: Other: Chapter Royalties; Atara: Consultancy; Sigms Tau: Research Funding. Logan:Jazz Pharmaceuticals: Consultancy; Amgen: Consultancy; Pharmacyclics: Consultancy. Martin:Sanofi: Consultancy.

Systemic Resistance to Powdery Mildew in Brassica napus (AACC) and Raphanus alboglabra (RRCC) by Trichoderma harzianum TH12.

Trichoderma harzianum TH12 is a microbial pesticide for certain rapeseed diseases. The mechanism of systemic resistance induced by TH12 or its cell-free culture filtrate (CF) in Brassica napus (AACC) and Raphanus alboglabra (RRCC) to powdery mildew disease caused by ascomycete Erysiphe cruciferarum was investigated. In this study, we conducted the first large-scale global study on the cellular and molecular aspects of B. napus and R. alboglabra infected with E. cruciferarum. The histological study showed the resistance of R. alboglabra to powdery mildew disease. The growth of fungal colonies was not observed on R. alboglabra leaves at 1, 2, 4, 6, 8, and 10 days post-inoculation (dpi), whereas this was clearly observed on B. napus leaves after 6 dpi. In addition, the gene expression of six plant defense-related genes, namely, PR-1, PR-2 (a marker for SA signaling), PR-3, PDF 1.2 (a marker for JA/ET signaling), CHI620, and CHI570, for both genotypes were analyzed in the leaves of B. napus and R. alboglabra after treatment with TH12 or CF and compared with the non-treated ones. The qRT-PCR results showed that the PR-1 and PR-2 expression levels increased in E. cruciferarum-infected leaves, but decreased in the TH12-treated leaves compared with leaves treated with CF. The expression levels of PR-3 and PDF1.2 decreased in plants infected by E. cruciferarum. However, expression levels increased when the leaves were treated with TH12. For the first time, we disclosed the nature of gene expression in B. napus and R. alboglabra to explore the resistance pathways in the leaves of both genotypes infected and non-infected by powdery mildew and inoculated or non-inoculated with elicitor factors. Results suggested that R. alboglabra exhibited resistance to powdery mildew disease, and the application of T. harzianum and its CF are a useful tool to facilitate new protection methods for resist or susceptible plants.

RTP1 encodes a novel endoplasmic reticulum (ER)-localized protein in Arabidopsis and negatively regulates resistance against biotrophic pathogens.

Oomycete pathogens cause serious damage to a wide spectrum of plants. Although host pathogen recognition via pathogen effectors and cognate plant resistance proteins is well established, the genetic basis of host factors that mediate plant susceptibility to oomycete pathogens is relatively unexplored. Here, we report on RTP1, a nodulin-related MtN21 family gene in Arabidopsis that mediates susceptibility to Phytophthora parasitica. RTP1 was identified by screening a T-DNA insertion mutant population and encoded an endoplasmic reticulum (ER)-localized protein. Overexpression of RTP1 rendered Arabidopsis more susceptible, whereas RNA silencing of RTP1 led to enhanced resistance to P.parasitica. Moreover, an RTP1 mutant, rtp1-1, displayed localized cell death, increased reactive oxygen species (ROS) production and accelerated PR1 expression, compared to the wild-type Col-0, in response to P.parasitica infection. rtp1-1 showed a similar disease response to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000, including increased disease resistance, cell death and ROS production. Furthermore, rpt1-1 exhibited resistance to the fungal pathogen Golovinomyces cichoracearum, but not to the necrotrophic pathogen Botrytis cinerea. Taken together, these results suggest that RTP1 negatively regulates plant resistance to biotrophic pathogens, possibly by regulating ROS production, cell death progression and PR1 expression.

CDPK1, an Arabidopsis thaliana calcium-dependent protein kinase, is involved in plant defense response

Growing evidences indicate that calcium-dependent protein kinases (CDPKs) are involved in many aspects of plant’s growth, development and responses to biotic and abiotic stresses. However, the biological roles of most CDPKs remain unclear. Here we report that CDPK1 is induced by infection with both virulent and avirulent Pseudomonas syringae strains. To understand the biological function of CDPK1, we analyzed phenotypes of the T-DNA insertion line (cpk10) which did not express the CDPK1 gene. The cpk10 mutant exhibited deduced expression of the defense-related genes PR1, PR2, and AIG1 following inoculation with the avirulent pathogen Pst AvrRpt2. In addition, the CDPK1 gene was found to be expressed in guard cells and the cpk10 mutant showed alteration in stomata closure after Pst DC3000 infection. We also found that expression of CDPK1 is induced by plant hormones salicylic acid, methyl jasmonate, and abscisic acid. These results indicate that CDPK1 may be involved in plant defense response.

Gaseous 3-pentanol primes plant immunity against a bacterial speck pathogen, Pseudomonas syringae pv. tomato via salicylic acid and jasmonic acid-dependent signaling pathways in Arabidopsis.

3-Pentanol is an active organic compound produced by plants and is a component of emitted insect sex pheromones. A previous study reported that drench application of 3-pentanol elicited plant immunity against microbial pathogens and an insect pest in crop plants. Here, we evaluated whether 3-pentanol and the derivatives 1-pentanol and 2-pentanol induced plant systemic resistance using the in vitro I-plate system. Exposure of Arabidopsis seedlings to 10 μM and 100 nM 3-pentanol evaporate elicited an immune response to Pseudomonas syringae pv. tomato DC3000. We performed quantitative real-time PCR to investigate the 3-pentanol-mediated Arabidopsis immune responses by determining Pathogenesis-Related (PR) gene expression levels associated with defense signaling through salicylic acid (SA), jasmonic acid (JA), and ethylene signaling pathways. The results show that exposure to 3-pentanol and subsequent pathogen challenge upregulated PDF1.2 and PR1 expression. Selected Arabidopsis mutants confirmed that the 3-pentanol-mediated immune response involved SA and JA signaling pathways and the NPR1 gene. Taken together, this study indicates that gaseous 3-pentanol triggers induced resistance in Arabidopsis by priming SA and JA signaling pathways. To our knowledge, this is the first report that a volatile compound of an insect sex pheromone triggers plant systemic resistance against a bacterial pathogen.

Induction of systemic resistance against Papaya ring spot virus (PRSV) and its vector Myzus persicae by Penicillium simplicissimum GP17-2 and silica (Sio2) nanopowder

The systemic expressions of certain defense-related genes in cucumber plants induced by culture filtrate (CF) of the plant growth-promoting fungus (PGPF), Penicillium simplicissimum GP17-2, and challenging with Sio2 nanoparticles against Papaya ring spot virus (PRSV) and its vector the green peach aphid Myzus persicae were investigated. CF and Sio2 were topically applied to cucumber plants by soil drench method. Results showed that the severity of PRSV and its accumulation in cucumber leaves were significantly reduced in all treated cucumber plants compared with the control. The expression of pathogenesis-related genes showed strong activations of peroxidases (POX) and phenylalanineammonia lyase (PAL) genes and moderate expression of PR-1 one day after inoculation, while, six days after inoculation a high activation of PAL and PR-1 genes and moderate activation of POX gene were detected. No expression was detected in plants infected with PRSV alone without any treatments. These results suggested that CF and Sio2 treatments mediated the expression of the majority of various pathogen-related genes, which may contribute to the increased defense mechanism against PRSV infection in cucumber.

Optimal level of purple acid phosphatase5 is required for maintaining complete resistance to Pseudomonas syringae.

Plants possess an exceedingly complex innate immune system to defend against most pathogens. However, a relative proportion of the pathogens overcome host's innate immunity and impair plant growth and productivity. We previously showed that mutation in purple acid phosphatase (PAP5) lead to enhanced susceptibility of Arabidopsis to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). Here, we report that an optimal level of PAP5 is crucial for mounting complete basal resistance. Overexpression of PAP5 impaired ICS1, PR1 expression and salicylic acid (SA) accumulation similar to pap5 knockout mutant plants. Moreover, plant overexpressing PAP5 was impaired in H2O2 accumulation in response to Pst DC3000. PAP5 is localized in to peroxisomes, a known site of generation of reactive oxygen species for activation of defense responses. Taken together, our results demonstrate that optimal levels of PAP5 is required for mounting resistance against Pst DC3000 as both knockout and overexpression of PAP5 lead to compromised basal resistance.

Abstract 4049: Neutrophil elastase induces post-transcriptional increase of surface MHC class I expression on lung cancer cells

Abstract Neutrophil infiltration and the presence of the neutrophil-associated serine protease neutrophil elastase (NE) is a poor prognosis factor for lung cancer patients. NE protein contains the nonomeric epitope for PR1, an immunogenic leukemia associated antigen recognized by a circulating population of functional cytotoxic T lymphocytes (CTL) in healthy individuals. As previously reported by our laboratory, solid tumor cells such as breast cancer cells internalize NE and cross-present PR1 in the context of the CTL ligand, MHC class I molecules, specifically HLA-A2. Internalization of NE by lung cancer cell lines has previously been reported by other groups. Therefore, we hypothesized that lung cancer cells internalize NE and cross-present PR1 on HLA-A2. A panel of lung cancer cell lines did not endogenously express proteins containing the PR1 epitope, NE and proteinase 3, at the transcript or protein level, using RT-PCR, western blot analysis and flow cytometry. Intracellular staining of NE was detected in lung cancer cell lines by flow cytometry after pulsing overnight with 10 μg/mL purified soluble NE. Subsequently, elevated surface expression of PR1:HLA-A2 complexes was determined using the PR1:HLA-A2-specific antibody generated within our laboratory (A. Sergeeva, et al., Blood 2011). Furthermore, an enhancement of HLA-A2 (antibody clone BB7.2) and pan-HLA-I (antibody clone W6/32) was observed. Through RT-PCR analysis, MHC class I genes (heavy chains HLA-A, -B, -C and the β2-microglobulin light chain) were not altered by NE exposure. Additionally, genes associated with the antigen processing machinery remained unchanged (tapasin, calnexin, calreticulin, and the Transporter associated with Antigen Processing 1 and 2). Thus exposure of lung cancer cells to NE enhances surface MHC class I molecules beyond the transcript level. MHC class I molecules serve as ligands for the T cell receptor of cytotoxic T lymphocytes, which can cause direct lysis of the target cell presenting a foreign or tumor-associated antigen. In these studies, we have detected enhanced expression of surface MHC class I molecules on lung cancer cells after exposure to NE. Thus these studies indicate that immunotherapies augmenting CTL activation could have the greatest efficacy for the poor prognosis lung cancer patients with high neutrophil infiltration or elevated plasma NE. NE contains the immunogenic PR1 epitope, and our studies have detected its cross-presentation on lung cancer cells. Therefore, anti-PR1 immunotherapies that have been developed for treatment of leukemia and lymphoma, could seamlessly be translated into lung cancer patients, and remains an area of active investigation in our laboratory. Citation Format: Haley L. Peters, Satyendra C. Tripathi, Hiro Katayama, Celine Kerros, Alex Perakis, Lisa S. St. John, Gheath Al-Atrash, Samir Hanash, Jeffrey J. Molldrem. Neutrophil elastase induces post-transcriptional increase of surface MHC class I expression on lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4049. doi:10.1158/1538-7445.AM2015-4049

Effect of the probiotic Lactobacillus rhamnosus on the expression of genes involved in European eel spermatogenesis

Positive effects of probiotics on fish reproduction have been reported in several species. In the present study, 40 male European eels were weekly treated with recombinant hCG for 9 weeks and with three different concentrations (103, 105, and 106 CFU/mL) of probiotic Lactobacillus rhamnosus IMC 501 (Sinbyotec, Italy). The probiotics were daily added to the water from the sixth week of the hCG treatment. Males from the treated and control groups were sacrificed after 1, 2, and 3 weeks of probiotic treatment (seventh–ninth weeks of hCG treatment); at this point, sperm and testis samples were also collected. Sperm volume was estimated, and motility was analyzed by computer-assisted sperm analysis software. Alternations in transcription of specific genes involved in reproductive process such as activin, androgen receptors α and β (arα and arβ), progesterone receptor 1 (pr1), bone morphogenetic protein 15 (bmp15), and FSH receptor (fshr) were analyzed in the testis. After 2 weeks of probiotic treatment, sperm production and sperm motility parameters (percentage of motile cells and percentage of straight-swimming spermatozoa) were increased in the European eel treated with 105 CFU/mL compared to controls or to the other probiotic doses. These changes were associated with increases in messenger RNA expression of activin, arα, arβ, pr1, and fshr. Conversely, after 3 weeks, activin and pr1 expression decreased. No significant changes were observed on bmp15 expression throughout the duration of the treatment with 105 CFU/mL dose. The lowest and highest probiotic dose (103 and 106 CFU/mL, respectively) inhibited the transcription of all genes along all the experiment, except for arα and arβ after 1 week of probiotic treatment when compared to controls. The changes observed by transcriptomic analysis and the sperm parameters suggest that a treatment with L rhamnosus at 105 CFU/mL for 2 weeks could improve spermatogenesis process in Anguilla anguilla.

Effects of light intensity on the susceptibility of Nicotiana tabacum to cucumber mosaic virus

Disease development in Nicotiana tabacum after inoculation with cucumber mosaic virus (CMV) was accelerated when plants were grown in high light (300 µmol m−2 s−1, HL) compared with those grown in low light (70 µmol m−2 s−1, LL). Several characteristic defense reactions, including release of reactive oxygen species, accumulation of salicylic acid (SA), expression of SA-mediated pathogenesis related (PR) proteins and the systemic development of disease, were proved to be light dependent. Plants exposed to HL or medium light (150 µmol m−2 s−1, ML) had serious oxidative damage caused by CMV infection compared with LL-plants. In addition, the levels of SA and mRNA transcripts for non-expressor of pathogenesis-related gene 1 in CMV-inoculated plants were the highest under LL with decreasing photon flux density. In HL-exposed plants, the expression of PR1 and PR2 were repressed, but the expression of PR5 was not significantly altered, revealing a complex cross talk between light intensity and the SA signaling pathway in regulating N. tabacum response to the infection of CMV. Taken together, our results revealed that increased light intensity reduced the resistance ability of N. tabacum to CMV, correlating with repression of the SA-dependent signaling pathway and a change in the cellular redox status in plants cells.

Functionalized mesoporous silica nanoparticles with redox-responsive short-chain gatekeepers for agrochemical delivery.

The controlled release of salicylic acid (SA), a key phytohormone, was mediated by using a novel decanethiol gatekeeper system grafted onto mesoporous silica nanoparticles (MSNs). The decanethiol was conjugated only to the external surfaces of the MSNs through glutathione (GSH)-cleavable disulfide linkages and the introduction of a process to assemble gatekeepers only on the outer surface so that the mesopore area can be maintained for high cargo loading. Raman and nitrogen sorption isotherm analyses confirmed the successful linkage of decanethiol to the surface of MSNs. The in vitro release of SA from decanethiol gated MSNs indicated that the release rate of SA in an environment with a certain amount of GSH was significantly higher than that without GSH. More importantly, in planta experiments showed the release of SA from decanethiol gated MSNs by GSH induced sustained expression of the plant defense gene PR-1 up to 7 days after introduction, while free SA caused an early peak in PR-1 expression which steadily decreased after 3 days. This study demonstrates the redox-responsive release of a phytohormone in vitro and also indicates the potential use of MSNs in planta as a controlled agrochemical delivery system.

The synthetic cationic lipid diC14 activates a sector of the Arabidopsis defence network requiring endogenous signalling components.

Natural and synthetic elicitors have contributed significantly to the study of plant immunity. Pathogen-derived proteins and carbohydrates that bind to immune receptors, allow the fine dissection of certain defence pathways. Lipids of a different nature that act as defence elicitors, have also been studied, but their specific effects have been less well characterized, and their receptors have not been identified. In animal cells, nanoliposomes of the synthetic cationic lipid 3-tetradecylamino-tert-butyl-N-tetradecylpropionamidine (diC14) activate the TLR4-dependent immune cascade. Here, we have investigated whether this lipid induces Arabidopsis defence responses. At the local level, diC14 activated early and late defence gene markers (FRK1, WRKY29, ICS1 and PR1), acting in a dose-dependent manner. This lipid induced the salicylic acid (SA)-dependent, but not jasmonic acid (JA)-dependent, pathway and protected plants against Pseudomonas syringae pv. tomato (Pst), but not Botrytis cinerea. diC14 was not toxic to plant or pathogen, and potentiated pathogen-induced callose deposition. At the systemic level, diC14 induced PR1 expression and conferred resistance against Pst. diC14-induced defence responses required the signalling protein EDS1, but not NDR1. Curiously, the lipid-induced defence gene expression was lower in the fls2/efr/cerk1 triple mutant, but still unchanged in the single mutants. The amidine headgroup and chain length were important for its activity. Given the robustness of the responses triggered by diC14, its specific action on a defence pathway and the requirement for well-known defence components, this synthetic lipid is emerging as a useful tool to investigate the initial events involved in plant innate immunity.

Salicylic acid and cysteine contribute to arbutin-induced alleviation of angular leaf spot disease development in cucumber

Arbutin induced suppression of angular leaf spot disease in cucumber resulting from lower populations of Pseudomonas syringae pv lachrymans in the infected tissues. This study provides insight into mechanisms that may potentially account for this effect. In the absence of the pathogen, exogenous arbutin-induced expression of PR1, the marker of salicylic acid signaling, increased the content of salicylic acid and modulated the cysteine pool. This suggested that arbutin promoted cucumber plants to a “primed” state. When challenged with the pathogen, the arbutin-treated plants showed strongly reduced infection symptoms 7 days after inoculation. At this time point, they were characterized by higher contents of free and protein-bound cysteine due to higher cysteine biosynthetic capacity related to increased activities of serine acetyltransferase and cysteine synthase when compared with plants infected without arbutin treatment. Moreover, in the arbutin-treated and infected plants the contents of free salicylic acid and its conjugates were also increased, partly owing to its biosynthesis via the phenylpropanoid pathway. We suggest that arbutin-induced abrogation of angular leaf spot disease in cucumber could be mediated by salicylic acid and cysteine-based signaling.

A CC-NBS-LRR type gene GHNTR1 confers resistance to southern root-knot nematode in Nicotiana.benthamiana and Nicotiana.tabacum

Root-knot nematodes are obligate parasites that invade the roots of agricultural plants and induce the formation of specialized feeding structures, especially races 3 and 4 of the southern root-knot nematode. However, not much is known about the defense mechanisms of plants against the invasion of M. incognita race 1. In this study, we characterized and performed functional analysis of the CC-NBS-LRR domain gene, GHNTR1. Using the GHNTR1 promoter to drive the GUS marker gene, we found that GUS expression was high in the roots and shoots of seedlings, four leaves stages, and mature stages. When GHNTR1 was transiently expressed in Nicotiana. benthamiana and Nicotiana. tabacum, necrosis was observed in the leaves and detectable amounts of H2O2 had accumulated when compared to the control plants. Stable transformation of N. benthamiana with the GHNTR1 gene using Agrobacterium induced the expression of defense marker genes PR1, PR2, LOX, and ERF1. In addition, the transgenic N. benthamiana and N. tabacum plants exhibited higher resistance to M. incognita infection. When transgenic N. tabacum were challenged with M. incognita, callose deposition and peroxide accumulation were observed in the roots by aniline-blue and DAB staining, respectively. Sequencing and bioinformatic analysis of small RNA revealed 48 miRNAs were up-regulated and 32 miRNAs were down-regulated. Further, 345 genes were predicted as targets of miRNAs and these included genes that are involved in cell death, death, and stress response. Thus, GHNTR1 is the first gene isolated from cotton that confers resistance against M. incognita and it induced a series of hypersensitive responses in transgenic tobacco. These findings improve our understanding of the molecular mechanisms of the response of upland cotton to M. incognita infection.

Expression of pathogenesis-related genes in Metarhizium anisopliae when infecting Spodoptera exigua

Characterization of pathogenesis genes of Metarhizium anisopliae, will provide better understanding of the role of these genes during pathogenesis. The expression profiles of pathogenesis-related genes encoding for a subtilisin-like protease (PR1), two types of chitinases (CHI2 and CHI3), and a peptide synthetase (PES) were studied during the different stages of M. anisopliae infection in Spodoptera exigua larvae using quantitative real-time RT-PCR. Sampling were at 0, 2, 12, and 24h after infection, when the infected larvae reached the moribund stage (36h), when mycelia emerged from the cadavers, when few conidia had formed on the mycelia, and when the cadavers were covered by conidia. For comparison, conidia and mycelial samples harvested from culture media were also included. Among the studied genes, PR1 expression was detected early at 2h after infection and increased as the infection progressed. CHI2 and CHI3 expressions were detected 12h after infection and when the mycelia emerged from cadavers, respectively. The expression levels of PR1, CHI2 and CHI3 genes increased significantly at the beginning of conidiogenesis on cadavers, but decreased at later stages. As expected, their expressions in pure fungal propagules were at very low levels. For PES gene, fold changes were not significant between different samples (less than onefold), indicating it might not have a major role in infecting stages. High expression levels of PR1, CHI2, and CHI3 genes during the post-mortem hyphal growth and conidiation stages of M. anisopliae clearly indicate the importance of these genes during the saprophytic phase of this fungus on host insect.

The influence of conidial Pr1 protease on pathogenicity potential of Metarhizium anisopliae senso latu to ticks.

Pr1 is a subtilisin-like protease produced by Metarhizium spp. entomopathogenic fungi, and it is recognized as heavily involved in the initial steps of the fungal invasion of arthropod-host cuticles. In the current study, correlation was sought between mortality of tick larvae and conidial Pr1 levels of one Metarhizium anisopliae senso latu (s.l.) isolate (CG 148). Conidia with different levels of pr1 gene expression and enzymatic activity were obtained by producing them on either artificial medium (to yield low Pr1 activity) or on Rhipicephalus microplus cadavers (to yield high Pr1 activity). Conidial proteolytic activity was assessed using N-suc-ala-ala-pro-phe-ρNA as the chromogenic substrate, and pr1 expression was profiled by qPCR using three genes (gpd, try, and tef) as reference genes. Pr1 enzymatic (proteolytic) activity on conidia obtained from tick cadavers was 36 U mg(-1) in comparison to 4 U mg(-1) on conidia from PDA medium. Also, pr1 gene expression level was ten times higher in conidia from tick cadavers compared to PDA medium. Bioassays of M. anisopliae s.l. CG 148 spores with elevated Pr1 proteolytic activity and gene expression levels did not demonstrate increased virulence (= significant change percent mortality of tick larvae). The minimal levels of Pr1 on conidia produced on artificial medium was adequate to afford high levels of virulence, and the elevated amounts of the enzyme on tick-cadaver-produced conidia did not induce elevated larval mortality. As long as some Pr1 activity was present, fungal virulence of isolate CG 148 against tick larvae was not elevated by increased levels of conidial Pr1.