A fungal protein elicitor PevD1 induces Verticillium wilt resistance in cotton

Key message

We found that the elicitor PevD1 triggered innate immunity in cotton, which plays an important role in future cotton wilt disease control.

Abstract

Elicitors can induce defense responses in plants and improve pathogen resistance. PevD1 is a secreted protein from Verticillium dahliae and activates the hypersensitive response and systemic acquired resistance to tobacco mosaic virus in tobacco plants. To investigate the PevD1-induced disease resistance mechanisms in cotton (Gossypium hirsutum), we report that Escherichia coli expressing PevD1 enhanced cotton resistance and the defense response to the fungal pathogen V. dahliae. The results showed that recombinant PevD1 improved cotton resistance when infiltrated at a concentration as low as 4 μg ml^?1, and the highest disease reduction was 38.16 % on the 15th day post V. dahliae inoculation. This protein was able to systemically induce hydrogen peroxide production, nitric oxide generation, lignin deposition, vessel reinforcement and defense enzymes, including phenylalanine ammonia-lyase, peroxidase, and polyphenol oxidase. PevD1 also enhanced the expression of three pathogenesis-related genes, namely, β-1,3-glucanase, chitinase, and cadinene synthase, and three key genes, PAL, C4H1, and 4CL, from the cotton defense phenylpropanoid metabolism pathway. Our results demonstrated that PevD1 acted as an effector in cotton and V. dahliae interactions and triggered innate immunity in cotton, resulting in the upregulation of defense-related genes, metabolic substance deposition and cell wall modifications. PevD1 is a candidate plant defense activator for cotton wilt disease control.     

Purification and expression of a protein elicitor from Alternaria tenuissima and elicitor-mediated defence responses in tobacco

A new protein elicitor, PeaT1, was purified from the mycelium of Alternaria tenuissima by column chromatography. PeaT1 was identified as a heat-stable and acidic protein. It induced systemic acquired resistance to tobacco mosaic virus (TMV) in tobacco plants but did not cause hypersensitive response. The elicitor-encoding gene was cloned by rapid amplification of cDNA ends method. Sequence analysis revealed that the cDNA is 624 bp in length and the open reading frame encodes for a polypeptide of 207 amino acids with a nascent polypeptide-associated complex domain. The peaT1 gene was cloned into the expression vector pET-28a and transformed into Escherichia coli BL21 (DE3). The recombinant elicitor also triggered defence responses in intact tobacco plants. The availability of the pure protein offers the possibility to isolate the corresponding receptor and links it to the downstream signalling pathway.     

PeaT1-induced systemic acquired resistance in tobacco follows salicylic acid-dependent pathway

Systemic acquired resistance (SAR) is an inducible defense mechanism which plays a central role in protecting plants from pathogen attack. A new elicitor, PeaT1 from Alternaria tenuissima, was expressed in Escherichia coil and characterized with systemic acquired resistance to tobacco mosaic virus (TMV). PeaT1-treated plants exhibited enhanced systemic resistance with a significant reduction in number and size of TMV lesions on wild tobacco leaves as compared with control. The quantitative analysis of TMV CP gene expression with real-time quantitative PCR showed there was reduction in TMV virus concentration after PeaT1 treatment. Similarly, peroxidase (POD) activity and lignin increased significantly after PeaT1 treatment. The real-time quantitative PCR revealed that PeaT1 also induced the systemic accumulation of pathogenesis-related gene, PR-1a and PR-1b which are the markers of systemic acquired resistance (SAR), NPR1 gene for salicylic acid (SA) signal transduction pathway and PAL gene for SA synthesis. The accumulation of SA and the failure in development of similar level of resistance as in wild type tobacco plants in PeaT1 treated nahG transgenic tobacco plants indicated that PeaT1-induced resistance depended on SA accumulation. The present work suggested that the molecular mechanism of PeaT1 inducing disease resistance in tobacco was likely through the systemic acquired resistance pathway mediated by salicylic acid and the NPR1 gene.     

The coat protein of potato virus X is a strain-specific elicitor of Rx1-mediated virus resistance in potato

The Rx1 gene in potato confers extreme resistance to potato virus X (PVX). To investigate the mechanism and elicitation of Rx resistance, protoplasts of potato cv. Cara (Rx1 genotype) and Maris Bard (rx1 genotype) were inoculated with PVX and tobacco mosaic virus (TMV). At 24 h post-inoculation in Maris Bard protoplasts there was at least 100-fold more PVX RNA than in protoplasts of Cara. TMV RNA accumulated to the same level in both types of protoplast. However, when the TMV was inoculated together with PVX the accumulation of TMV RNA was suppressed in the Cara (Rx1 genotype) protoplasts to the same extent as PVX. The Rx1 resistance also suppressed accumulation of a recombinant TMV in which the coat protein gene was replaced with the coat protein gene of PVX. It is therefore concluded that Rx1-mediated resistance is elicited by the PVX coat protein, independently of any other proteins encoded by PVX. The domain of the coat protein with elicitor activity was localized by deletion and mutation analysis to the structural core of a non-virion form of the coat protein.     

Stable isotope labelled mass spectrometry for quantification of the relative abundances for expressed proteins induced by PeaT1

The protein elicitor from the mycelium of Alternaria tenuissima has been isolated. The elicitor triggered resistance to the tobacco mosaic virus in tobacco by inducing relative oxygen species, but without causing hypersensitive necrosis. The elicitor is reported to impart resistance against Verticillium dahliae and to increase yield in cotton, but its mechanism is not yet clear. In this study, the stable isotope labelled mass spectrometry method was used to quantify the relative abundances of protein expression induced by PeaT1 in Arabidopsis. A significant difference in the relative abundances for the expression of different proteins related to metabolism, modification, regulatory, defense, stress and antioxidation was found in Arabidopsis.     

Effect of mitochondria-targeted antioxidant SkQ1 on programmed cell death induced by viral proteins in tobacco plants

Programmed cell death (PCD) is the main defense mechanism in plants to fight various pathogens including viruses. The best-studied example of virus-induced PCD in plants is Tobacco mosaic virus (TMV)-elicited hypersensitive response in tobacco plants containing the N resistance gene. It was previously reported that the animal mitochondrial protein Bcl-xL, which lacks a homolog in plants, effectively suppresses plant PCD induced by TMV p50 — the elicitor of hyper-sensitive response in Nicotiana tabacum carrying the N gene. Our studies show that the mitochondria-targeted antioxidant SkQ1 effectively suppresses p50-induced PCD in tobacco plants. On the other hand, SkQ1 did not affect Poa semilatent virus TGB3-induced endoplasmic reticulum stress, which is followed by PCD, in Nicotiana benthamiana epidermal cells. These data suggest that mitochondria-targeted antioxidant SkQ1 can be used to study molecular mechanisms of PCD suppression in plants.     

Analysis ofcis-regulatory elements involved in induction of a tobacco PR-5 gene by virus infection

cis-Regulatory elements involved in tobacco mosaic virus (TMV)-inducible expression were indentified in a tobacco PR-5 gene, encoding an acidic thaumatin-like protein. By fusing upstream sequences of the PR-5 gene to the GUS reporter gene and analysing transgenic plants containing these fusions for local and systemic induction of GUS activity by TMV, it was found that sequences between-1364 and-718 are involved in TMV induction of PR-5 gene expression.     

<Emphasis Type="SmallCaps">l</Emphasis>-Alanine augments rhizobacteria-induced systemic resistance in cucumber

Bacillus vallismortis strain EXTN-1 is a proven biotic elicitor of systemic resistance in many crops against various pathogens. l-Alanine (Ala) was tested in cucumber as a chemical elicitor of induced systemic resistance (ISR) against Colletotrichum orbiculare. In the greenhouse, both Ala and EXTN-1 induced significant levels of disease suppression in cucumber against anthracnose. When cucumber plants were treated with EXTN-1 and Ala together, augmentative disease suppression was observed. Experiments with transgenic tobacco plants carrying pathogenesis-related genes fused with the β-glucuronidase (GUS) reported gene (PR-1a::GUS & PDF 1.2::GUS) showed an enhanced activation of both PR-1a and PDF 1.2 genes upon combined treatment with Ala and EXTN-1. RT-PCR analysis with transgenic (PR-1a or PDF 1.2 over expressing) Arabidopsis plant showed more enhanced expression of resistance genes PR-1a and PDF 1.2 upon combined treatment with Ala and EXTN-1 than either alone. An augmentative ISR effect, when the bacterial elicitor and chemical elicitor were combined together, was confirmed.     

A DREPP protein interacted with PeaT1 from <Emphasis Type="Italic">Alternaria tenuissima</Emphasis> and is involved in elicitor-induced disease resistance in <Emphasis Type="Italic">Nicotiana</Emphasis> plants

PeaT1 is a proteinaceous elicitor from fungal pathogen Alternaria tenuissima. Our previous research revealed that this elicitor could induce defense response and enhance disease resistance in various plants including Nicotiana plants. However, immune activation mechanisms whereby PeaT1 elicits defense response remain unclear. In this study, the association between elicitor protein PeaT1 and the plasma membrane was assessed using the FITC (Fluorescein isothiocyanate) labeling method. A PeaT1-interacting protein was isolated via ¹²⁵I-PeaT1 cross-linking and Far Western blot analyses, and designated PtBP1 (PeaT1 Binding Protein 1). From the data of Mass spectrometry (MS) and bioinformatics analysis, the 22 kDa plasma membrane protein PtBP1 was inferred to be a member of DREPP (developmentally regulated plasma membrane polypeptide) family that is induced in plants under stress conditions and might get involved in downstream signaling. For further verification of this association, Far Western blot, co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) analyses were performed, showing PtBP1 could bind with PeaT1 in vitro and in vivo. Virus-induced gene silencing (VIGS) analysis exhibited that PtBP1 silencing in Nicotiana benthamiana attenuated tobacco mosaic virus (TMV) resistance compared to the tobacco rattle virus (TRV) control after PeaT1 treatment.     

The symptom difference induced by <Emphasis Type="Italic">Tobacco mosaic virus</Emphasis> and <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> in tobacco plants containing the <Emphasis Type="Italic">N</Emphasis> gene is determined by movement protein gene

Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) are two closely related viruses in the genus Tobamovirus, but they induce obviously different sizes of necrotic lesions in tobacco plants containing the N gene. Comparison of the symptoms produced by TMV, ToMV and a chimaeric virus (T/OMP), in which the TMV movement protein (MP) gene was replaced by the ToMV MP gene, showed T/OMP caused necrotic lesions that were similar in size to those of ToMV in tobacco plants containing the N gene. The coat protein and MP of the three viruses accumulated in planta with similar levels, and the replication level of TMV and T/OMP in protoplasts also had no difference. Comparison of the activities of defense-related enzymes (PAL, POD and PPO) induced by the three viruses also showed that the variability of enzyme activity induced by T/OMP was similar to that induced by TMV, but different from that induced by ToMV. The results indicate that the size difference of necrotic lesions induced by TMV and ToMV in tobacco plants containing the N gene results from the functional difference of their MP genes.     

Protein composition of tobacco leaves with antiviral resistance induced by activators of defense responses and TMV infection

In tobacco (Nicotiana tabacum L.) plants of hypersensitive cv. Samsun NN, a capability of necrosis lesion formation and protein patterns were studied after induction of antiviral resistance by defense responses activators (DRA) (arachidonic acid, ubiquinone 50, and vitamin E) and by infection with tobacco mosaic virus (TMV). DRA and TMV improved both local and systemic leaf resistance to TMV. Native protein electrophoresis demonstrated differences in the composition of leaf proteins extracted under acidic and alkaline conditions. SDS-PAGE revealed proteins accumulated during the development of systemic antiviral resistance after lower leaf treatments with DRA and of local resistance induced by pretreatment with TMV. It was shown that various DRA affected protein patterns similarly, whereas TMV infection resulted in other changes. It is supposed that different pathways function in tobacco plants during induction of systemic resistance by DRA and TMV infection.     

Tobacco plants epigenetically suppressed in phenylalanine ammonia-lyase expression do not develop systemic acquired resistance in response to infection by tobacco mosaic virus

The response of tobacco (Nicotiana tabacum L. cv. Xanthinc) plants, epigenetically suppressed for phenylalanine ammonia-lyase (PAL) activity, was studied following infection by tobacco mosaic virus (TMV). These plants contain a bean PAL2 transgene in the sense orientation, and have reduced endogenous tobacco PAL mRNA and suppressed production of phenylpropanoid products. Lesions induced by TMV infection of PAL-suppressed plants are markedly different in appearance from those induced on control plants that have lost the bean transgene through segregation, with a reduced deposition of phenofics. However, they develop at the same rate as on control tobacco, and pathogenesis-related (PR) proteins are induced normally upon primary infection. The levels of free salicylic acid (SA) produced in primary inoculated leaves of PAL-suppressed plants are approximately fourfold lower than in control plants after 84 h, and a similar reduction is observed in systemic leaves. PR proteins are not induced in systemic leaves of PAL-suppressed plants, and secondary infection with TMV does not result in the restriction of lesion size and number seen in control plants undergoing systemic acquired resistance (SAR). In grafting experiments between wild-type and PAL-suppressed tobacco, the SAR response can be transmitted from a PAL-suppressed root-stock, but SAR is not observed if the scion is PAL-suppressed. This indicates that, even if SA is the systemic signal for establishment of SAR, the amount of pre-existing phenylpropanoid compounds in systemic leaves, or the ability to synthesize further phenylpropanoids in response to the systemic signal, may be important for the establishment of SAR. Treatment of PAL-suppressed plants with dichloro-isonicotinic acid (INA) induces PR protein expression and SAR against subsequent TMV infection. However, treatment with SA, while inducing PR proteins, only partially restores SAR, further suggesting that de novo synthesis of SA, and/or the presence or synthesis of other phenylpropanoids, is required for expression of resistance in systemic leaves.     

Lactones Produced through Thermal Oxidation of Higher Fatty Acids

Inhibitors of plant virus infection with systemic effects were found in the culture filtrates of Basidiomycetes such as Fomes fomentarius and Schizophyllum commune. These inhibitors were widely distributed in Agaricales and Polyporales. The inhibitors designated as BAS (Basidiomycete Antiviral Substance) were highly active against the mechanical transmission of tobacco mosaic virus (TMV). No toxic effect was observed on the host plants. BAS-F, a polysaccharide produced by F. fomentarius, almost completely inhibited infection, when BAS-F at 2 μg/ml was applied to the same surface of leaves of Xanthi-nc tobacco 24 h before TMV inoculation to the upper surface of the leaves, and 500/0 inhibition was shown when BAS-F at 10 μg/ml was applied to the under surface of leaves. BAS-F also induced systemic resistance to the non-treated leaves when it was applied to only one leaf of the plant. BAS-F also had similar effects against the infection of TMV on bell pepper and tomato plants.     

Systemic induction of a Phytolacca insularis antiviral protein gene by mechanical wounding, jasmonic acid, and abscisic acid

We have isolated a gene encoding a ribosome-inactivating protein (RIP) from Phytolacca insularis, designated as P. insularis antiviral protein 2 (PIP2). The PIP2 gene contained an open reading frame encoding a polypeptide of 315 amino acids. The deduced amino acid sequence of PIP2 was similar to those of other RIPs from Phytolacca plants. Recombinant PIP2 was expressed in Escherichia coli and was used to investigate its biological activities. Recombinant PIP2 inhibited protein synthesis in rabbit reticulocyte lysate by inactivating ribosomes through N-glycosidase activity. It also exhibited antiviral activity against tobacco mosaic virus (TMV). Expression of the PIP2 gene was developmentally regulated in leaves and roots of P. insularis. Furthermore, expression of the PIP2 gene was induced in leaves by mechanical wounding. The wound induction of the PIP2 gene was systemic. Expression of the PIP2 gene also increased in leaves in a systemic manner after treatment with jasmonic acid (JA) and abscisic acid (ABA), but not with salicylic acid (SA). These results imply that plants have employed the systemic synthesis of the defensive proteins to protect themselves more efficiently from infecting viruses.     

Production of the AVR9 elicitor from the fungal pathogen Cladosporium fulvum in transgenic tobacco and tomato plants

Three constructs were used to study the expression of the avirulence gene Avr9 from the fungal tomato pathogen Cladosporium fulvum in plants. They include pAVIR1, pAVIR2 and pAVIR21, encoding the wild-type AVR9 protein and two hybrid AVR9 proteins containing the signal sequences of the pathogenesis-related proteins PR-S and PR-1a, respectively. Transgenic tobacco plants obtained with the three constructs showed a normal phenotype and produced AVR9 elicitor with the same specific necrosis-inducing activity as the wild-type AVR9 elicitor produced in planta by isolates of C. fulvum containing the Avr9 gene. Level of expression was not correlated with number of T-DNA integrations, but plants homozygous for the Avr9 gene produced more elicitor protein than heterozygous plants. The amino acid sequence of the processed AVR9 peptide present in apoplastic fluid (AF) of pAVIR1 transformed plants producing the wild-type AVR9 elicitor was identical to that of the wild-type AVR9 peptide isolated from C. fulvum-infected tomato leaves. Transgenic Cf0 genotypes of tomato, obtained by transformation with construct pAVIR21, showed a normal phenotype. However, transgenic F1 plants expressing the Avr9 gene, obtained from crossing transgenic Cf0 genotypes with wild-type Cf9 genotypes, showed delayed growth, necrosis and complete plant death indicating that the AVR9 peptide produced in plants carrying the Cf9 gene is deleterious. The necrotic defence response observed in Cf9 genotypes expressing the Avr9 gene support the potential to apply avirulence genes in molecular resistance breeding.     

Induction of Systemic Resistance to Tobacco Powdery Mildew by Tobacco Mosaic Virus, Tobacco Necrosis Virus or Ethephon

Local infections of either TMV or TNV in tobacco plants cv. Havana 425 (hypersensitive to TMV) proved effective in inducing systemic resistance to subsequent inoculation with the powdery mildew fungus Erysiphe cichoracearum DC. The proportion of leaf surface invaded by this pathogen and the amount of conidia it produced were both significantly lower in virus inoculated plants than in non-inoculated controls. However, the decrease in sporulation rate was less regularly observed than the reduction in leaf area infected. TMV was more effective than TNV in protecting tobacco plants from powdery mildew. E. cichoracearum is thus added to the list of challenge pathogens to which TMV or TNV are known to induce resistance in the host plants. Necrotic lesions caused to the leaves by local treatment with Ethephon (an ethylene-releasing compound) also conferred to tobacco some degree of systemic resistance to the same fungal pathogen, more frequently visible as a reduction of leaf area invaded. The protection due to the Ethephon lesions was in present experiments less marked than that of TMV. No effects against subsequent powdery mildew infection were obtained when point freeze necrotic lesions were provoked on the plants.     

Constitutive expression of an inducible lipoxygenase in transgenic tobacco decreases susceptibility to Phytophthora parasitica var. nicotianae

Plant lipoxygenases (LOXs) are key enzymes involved in the generation of fatty acid derivatives, called oxylipins. In tobacco, LOX gene expression and activity are very low in healthy tissues and are highly enhanced in response to infection by Phytophthora parasitica nicotianae and to elicitor treatment. We previously showed, using antisense-LOX1 plants, that expression of the tobacco LOX1 gene is required for the race-cultivar specific resistance of tobacco to Phytophthora parasitica nicotianae. In order to investigate the effect of over-expressing a LOX gene on plant resistance, we transformed tobacco plants with the LOX1 coding sequence fused to the CaMV 35S promoter. Four transgenic lines with enhanced levels of LOX protein and specific activity over control plants were selected for further analysis. These plants were macroscopically indistinguishable from WT plants. Upon stem inoculation, the sense-LOX1 plants displayed a significantly decreased susceptibility to virulent races of Phytophthora parasitica nicotianae, stem lesions being 2- to 3-fold shorter in the transgenic lines than in WT plants. Using a root inoculation assay, the survival rate of sense-LOX1 seedlings was increased about 4-fold compared to their WT counterparts, with 60 to 80% of transgenic plants vs 15 to 20% of WT controls remaining healthy following inoculation with Phytophthora parasitica nicotianae. This is the first demonstration that the over-expression of a LOX gene is sufficient to reduce the susceptibility of a host plant to an oomycete pathogen.     

Cloning and characterization of the Gossypium hirsutum major latex protein gene and functional analysis in Arabidopsis thaliana

The major latex protein (MLP) gene in Gossypium hirsutum was cloned and designated Gh-MLP. Expression in cotton root was induced by salt stress and Verticillium dahliae toxin, and bioinformatic analysis showed that Gh-MLP encodes a 157-amino acid protein that is similar to members of the MLP subfamily in the Bet v 1 family. Although the structure of MLP is similar to Bet v 1 family proteins, the sequence identity to other subfamilies of Bet v 1 proteins is less than 20%. The Gh-MLP promoter contains potential cis-acting elements for response to salt stress and fungal elicitor. RT-PCR analysis showed that Gh-MLP expression was rapidly induced by NaCl and V. dahliae toxin, and induction was maintained over 72 h. However, Gh-MLP transgenic Arabidopsis thaliana did not show resistance to V. dahiae, salt tolerance was significantly enhanced. In contrast to the wild type, the Gh-MLP transgene allowed plants to germinate normally after treatment with 75 mM NaCl. Total flavonoid was twofold higher in transgenic Arabidopsis than in the control, suggesting that Gh-MLP might be involved in altering flavonoid content. We hypothesize Gh-MLP, like other Bet v 1 family proteins, participates in the binding or transport of ligands through its specific three-dimensional structure, and takes part in defensive responses to biotic and abiotic stresses.     

RNA-dependent RNA Polymerase Activities in Tomato Plants Susceptible or Resistant to Tobacco Mosaic Virus

RNA-dependent RNA polymerase activities were measured in healthyand tobacco mosaic virus (TMV)-infected tomato plants, to investigatethe possibility that altered activity might be involved in theoperation of the Tm-I gene for resistance to TMV. Healthy, susceptibleand resistant plants had similar levels of enzyme activity.Infection with TMV strain 0, which is inhibited by Tm-I, causeda 2-fold increase in activity in susceptible plants but no increasein Tm-I plants. Infection with a number of strain 1 isolates,which overcome Tm-I resistance, led to a 2 to 4-fold increasein enzyme activity in resistant plants. RNA-dependent RNA polymerase, Tm-I resistance gene, tobacco mosaic virus, tomato, Lycopersicon esculentum