Enhanced <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> immune responses caused by heterologous plant genes from <Emphasis Type="Italic">Pinellia ternata</Emphasis>

Background

Pinellia ternata is a Chinese traditional medicinal herb, used to cure diseases including insomnia, eclampsia and cervical carcinoma, for hundreds of years. Non-self-recognition in multicellular organisms can initiate the innate immunity to avoid the invasion of pathogens. A design for pathogen independent, heterosis based, fresh resistance can be generated in F₁ hybrid was proposed.

Results

By library functional screening, we found that P. ternata genes, named as ptHR375 and ptHR941, were identified with the potential to trigger a hypersensitive response in Nicotiana benthamiana. Significant induction of ROS and Callose deposition in N. benthamiana leaves along with activation of pathogenesis-related genes viz.; PR-1a, PR-5, PDF1.2, NPR1, PAL, RBOHB and ERF1 and antioxidant enzymes was observed. After transformation into N. benthamiana, expression of pathogenesis related genes was significantly up-regulated to generate high level of resistance against Phytophthora capsici without affecting the normal seed germination and morphological characters of the transformed N. benthamiana. UPLC-QTOF-MS analysis of ptHR375 transformed N. benthamiana revealed the induction of Oxytetracycline, Cuelure, Allantoin, Diethylstilbestrol and 1,2-Benzisothiazol-3(2H)-one as bioactive compounds. Here we also proved that F₁ hybrids, produced by crossing of the ptHR375 and ptHR941 transformed and non-transformed N. benthamiana, show significant high levels of PR-gene expressions and pathogen resistance.

Conclusions

Heterologous plant genes can activate disease resistance in another plant species and furthermore, by generating F₁ hybrids, fresh pathogen independent plant immunity can be obtained. It is also concluded that ptHR375 and ptHR941 play their role in SA and JA/ET defense pathways to activate the resistance against invading pathogens.

     

Linoleic acid-induced expression of defense genes and enzymes in tobacco

Linoleic acid (LA) is a naturally occurring fatty acid (FA) found to elicit induced systemic resistance (ISR) of tobacco against the bacterial soft rot pathogen, Pectobacterium carotovorum subsp. carotovorum (PCC). In this study, we examined effects of six doses of exogenous LA on the induction of defense genes and enzymes. The optimum ISR activity was observed in plants treated with 0.1 mM LA where the effect of LA on membrane permeability was minimal. The application of LA as a root drench enhanced the activity of defense enzymes such as phenylalanine ammonia-lyase (PAL), peroxidase (POD), and polyphenol oxidase (PPO) and induced the expression of β-glucuronidase (GUS). PAL and POD activities were increased in a concentration dependent manner while the maximum PPO activity was observed after treatment with 0.01 mM LA. An RT-PCR analysis of the defense-related genes, Coi1, NPR1, PR-1a and PR-1b, of tobacco plants treated with 0.1 mM LA revealed an association of LA with elicitation of ISR in tobacco.     

Induction of some defense-related genes and oxidative burst is required for the establishment of systemic acquired resistance in <Emphasis Type="Italic">Capsicum annuum</Emphasis>

The inoculation of primary pepper leaves with an avirulent strain of Xanthomonas campestris pv. vesicatoria induced systemic acquired resistance (SAR) in the non-inoculated, secondary leaves. This SAR response was accompanied by the systemic expression of the defense-related genes, a systemic microoxidative burst generating H₂O₂, and the systemic induction of both ion-leakage and callose deposition in the non-inoculated, secondary leaves. Some defense-related genes including those encoding PR-1, chitinase, osmotin, peroxidase, PR10, thionin, and SAR8.2 were markedly induced in the systemic leaves. The conspicuous systemic accumulation of H₂O₂ and the strong increase in peroxidase activity in the pepper leaves was suggested to play a role in the cell death process in the systemic micro-hypersensitive responses (HR), leading to the induction of the SAR. Treatment of the primary leaves with diphenylene iodinium (DPI), an inhibitor of oxidative burst, substantially reduced the induction of some of the defense-related genes, and lowered the activation of the oxidative bursts in the systemic leaves distant from the site of the avirulent pathogen inoculation and subsequently SAR. Overall, these results suggest that the induction of some defense-related genes as well as a rapid increase in oxidative burst is essential for establishing SAR in pepper plants.     

NtPR1a regulates resistance to Ralstonia solanacearum in Nicotiana tabacum via activating the defense-related genes

Pathogenesis-related proteins (PRs) are associated with the development of systemic acquired resistance (SAR) against further infection enforced by fungi, bacteria and viruses. PR1a is the first PR-1 member that could be purified and characterized. Previous studies have reported its role in plants’ resistance system against oomycete pathogens. However, the role of PR1a in Solanaceae plants against the bacterial wilt pathogen Ralstonia solanacearum remains unclear. To assess roles of NtPR1a in tobacco responding to R. solanacearum, we performed overexpression experiments in Yunyan 87 plants (a susceptible tobacco cultivar). The results illuminated that overexpression of NtPR1a contributed to improving resistance to R. solanacearum in tobacco Yunyan 87. Specifically speaking, NtPR1a gene could be induced by exogenous hormones like salicylic acid (SA) and pathogenic bacteria R. Solanacearum. Moreover, NtPR1a-overexpressing tobacco significantly reduced multiple of R. solanacearum and inhibited the development of disease symptoms compared with wild-type plants. Importantly, overexpression of NtPR1a activated a series of defense-related genes expression, including the hypersensitive response (HR)-associated genes NtHSR201 and NtHIN1, SA-, JA- and ET-associated genes NtPR2, NtCHN50, NtPR1b, NtEFE26, and Ntacc oxidase, and detoxification-associated gene NtGST1. In summary, our results suggested that NtPR1a-enhanced tobacco resistance to R. solanacearum may be mainly dependent on activation of the defense-related genes.     

SGT1b is required for HopZ3-mediated suppression of the epiphytic growth of Pseudomonas syringae on N. benthamiana

Type III secreted effectors shape the potential of bacterial pathogens to cause disease on plants. Some effectors affect pathogen growth only in specific niches. For example, HopZ3 causes reduced epiphytic growth of Pseudomonas syringae strain B728a on Nicotiana benthamiana. This raises the question of whether genes important for effector-triggered disease resistance are needed for responses to effectors whose major effect is in the epiphytic niche. We report that SGT1b, a protein known to be important for defense activation, is essential for HopZ3-mediated suppression of PsyB728a epiphytic growth. SGT1b is required for HopZ3- and AvrB3-induced cell death in N. benthamiana plants that express the Pto resistance gene from tomato. We suggest that HopZ3 activates R gene mediated responses in N. benthamiana.     

The Tomato Fni3 Lysine-63–Specific Ubiquitin-Conjugating Enzyme and Suv Ubiquitin E2 Variant Positively Regulate Plant Immunity

The activation of an immune response in tomato (Solanum lycopersicum) against Pseudomonas syringae relies on the recognition of E3 ligase–deficient forms of AvrPtoB by the host protein kinase, Fen. To investigate the mechanisms by which Fen-mediated immunity is regulated, we characterize in this study a Fen-interacting protein, Fni3, and its cofactor, S. lycoperiscum Uev (Suv). Fni3 encodes a homolog of the Ubc13-type ubiquitin-conjugating enzyme that catalyzes exclusively Lys-63–linked ubiquitination, whereas Suv is a ubiquitin-conjugating enzyme variant. The C-terminal region of Fen was necessary for interaction with Fni3, and this interaction was required for cell death triggered by overexpression of Fen in Nicotiana benthamiana leaves. Fni3 was shown to be an active E2 enzyme, but Suv displayed no ubiquitin-conjugating activity; Fni3 and Suv together directed Lys-63–linked ubiquitination. Decreased expression of Fni3, another tomato Ubc13 homolog, Sl-Ubc13-2, or Suv in N. benthamiana leaves diminished cell death associated with Fen-mediated immunity and cell death elicited by several other resistance (R) proteins and their cognate effectors. We also discovered that coexpression of Fen and other R proteins/effectors with a Fni3 mutant that is compromised for ubiquitin-conjugating activity diminished the cell death. These results suggest that Fni3/Sl-Ubc13-2 and Suv regulate the immune response mediated by Fen and other R proteins through Lys-63–linked ubiquitination.     

Implications of oligomeric forms of POD-1 and POD-2 proteins isolated from cell walls of the biocontrol agent Pythium oligandrum in relation to their ability to induce defense reactions in tomato

The cell wall protein fraction (CWP) isolated from the biocontrol agent Pythium oligandrum induces defense reactions in tomato. CWP contains two novel elicitin-like proteins, POD-1 and POD-2, both with seven cysteines. To determine the essential structure in the defense-eliciting components of CWP, five fractions (F1, F2, F3, F4 and F5) were fractionated from CWP using cation chromatography and their components and disulfide bond compositions were analyzed. The expression levels of three defense-related genes (PR-6, LeCAS and PR-2b) were determined in tomato roots treated with each of the five fractions. Of the five fractions, F4 containing a heterohexamer of POD-1 and POD-2, and F5 containing a homohexamer of POD-1, both with disulfide bonds formed between all cysteine residues, induced the expression of three genes. F4 treatment also induced the accumulation of ethylene in tomato. The predicted three-dimensional structures of POD-1 and POD-2, and the results of SEC and MALDI-TOF MS analyses suggest that F4 consists of three POD-1 and POD-2 disulfide-bonded heterodimers that interleave into a hexameric ring through noncovalent association. These results suggest that this structure, which F5 also appears to form, is essential for stimulating defense responses in tomato.     

The type III effector RipB from Ralstonia solanacearum RS1000 acts as a major avirulence factor in Nicotiana benthamiana and other Nicotiana species

Ralstonia solanacearum is the causal agent of bacterial wilt in solanaceous crops. This pathogen injects approximately 70 effector proteins into plant cells via the Hrp type III secretion system in an early stage of infection. To identify an as-yet-unidentified avirulence factor possessed by the Japanese tobacco-avirulent strain RS1000, we transiently expressed RS1000 effectors in Nicotiana benthamiana leaves and monitored their ability to induce effector-triggered immunity (ETI). The expression of RipB strongly induced the production of reactive oxygen species and the expressions of defence-related genes in N. benthamiana. The ripB mutant of RS1002, a nalixidic acid-resistant derivative of RS1000, caused wilting symptoms in N. benthamiana. A pathogenicity test using R. solanacearum mutants revealed that the two already known avirulence factors RipP1 and RipAA contribute in part to the avirulence of RS1002 in N. benthamiana. The Japanese tobacco-virulent strain BK1002 contains mutations in ripB and expresses a C-terminal-truncated RipB that lost the ability to induce ETI in N. benthamiana, indicating a fine-tuning of the pathogen effector repertoire to evade plant recognition. RipB shares homology with Xanthomonas XopQ, which is recognized by the resistance protein Roq1. The RipB-induced resistance against R. solanacearum was abolished in Roq1-silenced plants. These findings indicate that RipB acts as a major avirulence factor in N. benthamiana and that Roq1 is involved in the recognition of RipB.     

<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.     

Expression of the ginseng PgPR10-1 in Arabidopsis confers resistance against fungal and bacterial infection

Korean ginseng (Panax ginseng C. A. Meyer) consists of nine cultivars from three Jakyung, Chungkyung, and Hwangsook lines. Among three previously identified PR-10 homologs from ginseng (PgPR10-1, PgPR10-2, and PgPR10-3), we found that the exact same sequence of PgPR10-2 exist in all tested nine cultivars. But a deletion and SNP was found in American ginseng (Panax quinquefolius). PR-10 proteins are known to be small and cytosolic, and showed similar three-dimensional structure. Here we show that the heterologous overexpression of PgPR10-1 in Arabidopsis showed enhanced resistance against Pseudomonas syringe, Fusarium oxysporum, and Botrytis cinerea and in-frame tagging with fluorescent protein showed its cytoplasm and nucleus localization. Protein–protein interaction of PgPR10-2 with PgPR10-1, PgPR10-2 and PgPR10-3 suggests that the PgPR10 proteins might form multimeric complexes in different cellular compartments to function in development and in defense-related mechanism. Differential response of PgPR10-1 and PgPR10-2 against different sets of biotic stresses in ginseng plant supports this notion.     

The receptor-like kinase SERK3/BAK1 is required for basal resistance against the late blight pathogen phytophthora infestans in Nicotiana benthamiana

Background

The filamentous oomycete plant pathogen Phytophthora infestans causes late blight, an economically important disease, on members of the nightshade family (Solanaceae), such as the crop plants potato and tomato. The related plant Nicotiana benthamiana is a model system to study plant-pathogen interactions, and the susceptibility of N. benthamiana to Phytophthora species varies from susceptible to resistant. Little is known about the extent to which plant basal immunity, mediated by membrane receptors that recognise conserved pathogen-associated molecular patterns (PAMPs), contributes to P. infestans resistance.

Principal Findings

We found that different species of Phytophthora have varying degrees of virulence on N. benthamiana ranging from avirulence (incompatible interaction) to moderate virulence through to full aggressiveness. The leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1/SERK3 is a major modulator of PAMP-triggered immunity (PTI) in Arabidopsis thaliana and N. benthamiana. We cloned two NbSerk3 homologs, NbSerk3A and NbSerk3B, from N. benthamiana based on sequence similarity to the A. thaliana gene. N. benthamiana plants silenced for NbSerk3 showed markedly enhanced susceptibility to P. infestans infection but were not altered in resistance to Phytophthora mirabilis, a sister species of P. infestans that specializes on a different host plant. Furthermore, silencing of NbSerk3 reduced the cell death response triggered by the INF1, a secreted P. infestans protein with features of PAMPs.

Conclusions/Significance

We demonstrated that N. benthamiana NbSERK3 significantly contributes to resistance to P. infestans and regulates the immune responses triggered by the P. infestans PAMP protein INF1. In the future, the identification of novel surface receptors that associate with NbSERK3A and/or NbSERK3B should lead to the identification of new receptors that mediate recognition of oomycete PAMPs, such as INF1.     

SEC14 Phospholipid Transfer Protein Is Involved in Lipid Signaling-Mediated Plant Immune Responses in Nicotiana benthamiana

We previously identified a gene related to the SEC14-gene phospholipid transfer protein superfamily that is induced in Nicotiana benthamiana (NbSEC14) in response to infection with Ralstonia solanacearum. We here report that NbSEC14 plays a role in plant immune responses via phospholipid-turnover. NbSEC14-silencing compromised expression of defense–related PR-4 and accumulation of jasmonic acid (JA) and its derivative JA-Ile. Transient expression of NbSEC14 induced PR-4 gene expression. Activities of diacylglycerol kinase, phospholipase C and D, and the synthesis of diacylglycerol and phosphatidic acid elicited by avirulent R. solanacearum were reduced in NbSEC14-silenced plants. Accumulation of signaling lipids and activation of diacylglycerol kinase and phospholipases were enhanced by transient expression of NbSEC14. These results suggest that the NbSEC14 protein plays a role at the interface between lipid signaling-metabolism and plant innate immune responses.