EIN2-mediated signaling is involved in pre-invasion defense in Nicotiana benthamiana against potato late blight pathogen, Phytophthora infestans

ABSTRACT

Nicotiana benthamiana ABCG1 and ABCG2 are ABC transporters which are probably involved in the export of capsidiol, the major phytoalexin of Nicotiana species. While capsidiol export by these transporters plays an essential role in post-invasion defense against Phytophthora infestans, they also export unidentified antimicrobial compound(s) involved in pre-invasion defense. In this study, promoter activity of NbABCG2 (Pabcg2a) was analyzed using a GFP marker. Expression of GFP under the control of Pabcg2a was significantly increased by co-expression with the INF1 elicitor from P. infestans. Disruption of the ethylene-responsive GCC box in Pabcg2a compromised INF1-induced activation of Pabcg2a. Consistently, penetration by P. infestans was increased by gene-silencing of NbEIN2, the key ethylene-signaling component, suggesting the involvement of ethylene for pre-invasion defense of N. benthamiana.

Plants can produce a wide range of cytotoxic compounds to prevent infection by potential pathogens.¹ Constitutively produced antimicrobial metabolites, called phytoanticipins, can prevent infection attempts by microbes. Plant cells also have physical barriers, such as the cuticle covering leaf and stem surfaces and cell walls, that prevent penetration by pathogens.² Nicotiana benthamiana is a model solanaceous plant with resistance to the potato late blight pathogen, Phytophthora infestans.³ When the leaf surface of N. benthamiana is inoculated with a zoospore suspension of P. infestans, only a few of the penetration attempts by the pathogen are successful,³ indicating that pre-penetration defense plays an important role for the defense of N. benthamiana against P. infestans.

Through screening based on virus-induced gene silencing (VIGS), we previously isolated over 30 unique genes required for resistance of N. benthamiana against P. infestans,^4,5 and identified NbABCG1a, -1b, -2a and -2b (hereafter NbABCG1/2), which encode full size ABCG (PDR-type) transporters, as required genes for resistance of N. benthamiana against P. infestans.⁵ NbABCG1/2 are predicted as functionally redundant exporters of capsidiol,⁵ the major sesquiterpenoid phytoalexin produced by Nicotiana and Capsicum species. Capsidiol production in N. benthamiana leaves can be induced by treatment with INF1 elicitor (a secretory protein produced by P. infestans⁶), and when production is suppressed by gene-silencing of NbEAH, encoding the dedicated enzyme for capsidiol production (5-epi-aristolochene dihydroxylase), the resistance of N. benthamiana against P. infestans is compromised,³ indicating that capsidiol plays a major role in post-invasion defense in N. benthamiana. Because expression of NbABCG1/2 genes is significantly upregulated in N. benthamiana leaves treated with INF1 elicitor,⁵ it was expected that expression of enzyme genes for capsidiol production and genes for corresponding transporters are regulated by a common mechanism during the induction of disease resistance in N. benthamiana.

To further investigate the regulation of genes for these ABC transporters, we analyzed promoter activity of NbABCG2a (Pabcg2a) in this study. A plasmid vector containing the GFP gene under the control of the NbABCG2a promoter sequence, Pabcg2a (500-bp 5′-flanking fragment, Fig. 1A), was constructed (Table S1). Pabcg2a:GFP was introduced to N. benthamiana leaves via agroinfiltration, and promoter activity was assessed by fluorescence microscopy (see Supplemental data for details of the method). While weak expression of GFP was detected by introduction of Pabcg2a:GFP, GFP expression was greatly enhanced by co-expression of INF1 under the control of the CaMV 35S promoter (Fig. 1B). In the promoter sequence of NbABCG2a, a canonical ethylene-responsive element GCC box (AGCCGCC)⁷ was identified at position −180 to −174 (Fig. 1A). The GCC boxes were also found in the promoter sequences of NbABCG1a, -1b and -2b.⁵ Given that gene silencing of NbEIN2, the gene for a central regulator of ethylene signaling, reduced expression of NbABCG1 and NbABCG2⁵, GCC boxes found in the promoter sequence of NbABCG1/2 genes were expected to play an important role for regulation of NbABCG1/2 expression. A vector for GFP under the control of Pabcg2a containing G to T mutations in GCC box (i.e. ATCCTCC), designated Pabcg2a tcc, was constructed (Table S1). Disruption of the GCC box significantly reduced the expression of GFP (Fig. 1B), indicating that the GCC box is crucial for regulating NbABCG2a expression during plant defense. However, a 200-bp 5′-flanking fragment of Pabcg2a did not induce GFP expression, even though this 200-bp fragment includes the GCC box (Fig. 1A, C), suggesting that the GCC box is essential, but not sufficient, for INF1-induced activation of NbABCG2a promoter. It should be also noted that there could be cis-acting element outside of 500-bp Pabcg2a tested in this study, as previous study for analysis of promoter region of N. plumbaginifolia PDR1, the ortholog of NbABCG2, revealed that there is cis element between positions −827 and −802⁸.

Figure 1.

Analysis of NbABCG2a promoter activity in epidermal cells of Nicotiana benthamiana. (A) Nucleotide sequence of promoter region of NbABCG2a gene. The 7-bp sequence for the expected GCC box motif is in red letters, the start codon of NbABCG2a is in blue. Sequence used for the pNPP243-Pabcg2a 200:GPF construct is underlined. (B) Expression of GFP in epidermal cells of N. benthamiana after inoculation with A. tumefaciens containing the indicated promoter-GFP constructs with or without co-expression of elicitor INF1. GFP fluorescence was monitored at 48 h post inoculation (hpi). Bars = 100 µm. (C) GFP fluorescence in epidermal cells of N. benthamiana after inoculation with A. tumefaciens containing the indicated promoter-GFP constructs. Bars = 100 µm. (D) qRT-PCR analysis of GFP expression under the control of the NbABCG2a promoters in N. benthamiana leaves at 48 hpi. Data are means ± SE (n = 4). Asterisks indicate significant differences in the indicated comparisons in Student's 2-tail t-test at **P < 0.01.

Expression of GFP under the control of the wild type and mutated Pabcg2a was quantified using qRT-PCR (Fig. 1D). Forty-eight hours after Agrobacterium-mediated introduction of Pabcg2a:GFP or mutated Pabcg2a tcc:GFP in leaves of N. benthamiana, the expression level of GFP was scored using a co-expressed truncated GUS gene driven by the 35S promoter as an internal standard. As a result, co-expression of INF1 significantly induced transcription of Pabcg2a:GFP but not Pabcg2a tcc:GFP, supporting the GFP-fluorescence observations (Fig. 1D). Moreover, mutation of the GCC box reduced the basal expression level of GFP without co-expression of INF1 (Fig. 1D). Expression of endogenous NbABCG2 is upregulated by A. tumefaciens inoculation itself and further enhanced by INF1 expression (Fig. S1), thus indicating that GCC box is also involved in the induced expression of NbABCG2 by A. tumefaciens infection.

Production of capsidiol plays a major role in post-invasion defense of N. benthamiana against P. infestans.^3,5 Although NbABCG1/2 are probable exporters of capsidiol, disease symptoms caused by P. infestans on NbABCG1/2-silenced plant were significantly more severe than on NbEAS-silenced plants, in which the production of capsidiol and related phytoalexins was specifically compromised.^3,5,9 Gene silencing of NbABCG1/2, but not of NbEAS, resulted in increased penetration by P. infestans; thus, NbABCG1/2 are also expected to be involved in pre-invasion defense via the export of unidentified antimicrobial compound(s).⁵

To investigate the role of ethylene signaling on pre-invasion defense in N. benthamiana, we determined the penetration rate of P. infestans on leaves of NbABCG1/2-silenced and NbEIN2-sileinced plants. A zoosporangia suspension of P. infestans was drop-inoculated on N. benthamiana leaves, and callose accumulation beneath the attempted penetration sites of pathogen was detected as a bright spot by aniline blue staining 24 h after inoculation. Calcofluor white was also used to visualize the pathogen (Fig. 2A). Sites with callose accumulation under the germ tube from zoosporangia on the leaf surface of gene-silenced plants were counted as sites of attempted penetration. Both NbABCG1/2- and NbEIN2-silenced plants had more callose spots than in control plants, and there was no significant difference in penetration rate of the pathogen between NbABCG1/2- and NbEIN2-silenced plants (Fig. 2B). These results confirmed that NbABCG1/2 are involved in pre-invasion defense and also indicated that NbEIN2 is required for full pre-invasion defense of N. benthamiana, probably through the transcriptional regulation of NbABCG1/2 gene.

Figure 2.

NbABCG1/2 and NbEIN2 are involved in pre-invasion defense of N. benthamiana to P. infestans. (A) Penetration sites on epidermal cell of non-silenced (TRV) and silenced (TRV:ABCG1/2 and TRV:EIN2) N. benthamiana leave at 24 h after inoculation (hpi) as detected by callose accumulation stained with aniline blue. P. infestans was visualized by calcofluor white staining. Arrowheads indicate penetration sites of P. infestans. a, appressorium-like swelling; z, zoosporangium. Bars = 30 µm. (B) Number of penetration sites was counted on leaf discs of control or gene-silenced N. benthamiana 24 hpi. Data are means ± SE (n = 6). Asterisks indicate significant differences in the indicated comparisons in Student's 2-tail t-test at *P<0.05.

Previously, we showed that resistance in NbEIN2-silenced N. benthamiana against P. infestans was compromised.³ VIGS-based screening identified several genes encoding enzymes related to the production of ethylene, namely S-adenosylmethionine synthetase, cystathionine gamma-synthase, S-adenosylhomocysteine hydrolase and 3 amino cyclopropanecarboxylate oxidases, as required genes for resistance of N. benthamiana to P. infestans.⁵ In NbEIN2-silenced plants, expression of NbEAS and NbEAH genes is reduced and leads to significant reduction in capsidiol production.^{3, 9} Collectively, these data indicate that EIN2-mediated ethylene signaling plays a central role for the induction of capsidiol production. Thus, it is reasonable that increased production of the corresponding transporters (i.e., NbABCG1/2) is also under the control of ethylene signaling.

In this study, we showed that ethylene signaling is also involved in pre-invasion defense of N. benthamiana against P. infestans (Fig. 2). NtPDR1 and NpPDR1, probable orthologs of NbABCG1 and NbABCG2, respectively, are reported as the transporters of diterpenes, such as sclareol, manool and cembrene.^{10, 11} Sclareol, a constitutively produced diterpene with antifungal activity to several pathogens,¹² inhibited the germination of P. infestans zoosporangia in our study (Fig. S2) as described previously.¹³ Constitutive expression of NpPDR1 have been detected in leaf glandular trichomes,¹⁰ where active secretion of substances has been observed.¹⁴ Thus, the agent for pre-invasion defense of N. benthamiana could be diterpene(s) secreted via NbABCG1/2. Moreover, sclareol and ethylene can induce lignification of plant cell walls,¹⁵ and thus strengthen mechanical barriers to pathogen penetration.^{16, 17} Therefore, ethylene-mediated expression of NbABCG1/2 and transported diterpenes have the potential to act in several ways to reinforce preformed defense at the surface of plant cells against attacking plant pathogens (Fig. 3).

Figure 3.

Model for the role of ethylene in pre- and post-invasion defense of N. benthamiana against P. infestans. Adapted from Shibata et al. (2016).⁵ (Top) Pre-invasion defense. Constitutively produced diterpene(s) are secreted by Nb-ABCG1/2 in response to invasion by P. infestans and inhibit the fungus. Basal expression of NbABCG1/2 is enhanced by ethylene. Secreted diterpene(s) might also increase pre-invasion defense via enhanced production of lignin.¹² (Bottom) Post-invasion defense. Recognition of the invading pathogen increases ethylene production, which leads to enhanced expression of genes for capsidiol production and secretion. MEP; methylerythritol phosphate, MVA; mevalonate, IPP; isopentenyl pyrophosphate, DMAPP; dimethylallyl pyrophospate, FPP; farnesyl pyrophosphate, GGPP; geranylgeranyl pyrophosphate, EA; 5-epi-aristolochene.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgements

We thank Prof. David C. Baulcombe (University of Cambridge, USA) for providing pTV00 and pBINTRA6 vectors, Prof. Gregory B. Martin (Cornell University, USA) for access to the N. benthamiana genome database, Dr. Kayo Shirai (Hokkaido Central Agricultural Experiment Station, Japan) and Dr. Seishi Akino (Hokkaido University, Japan) for providing P. infestans isolate 08YD1 and Dr. David A. Jones (Australian National University) for providing N. benthamiana seeds. We also thank Dr. Beth E. Hazen for English editing of the manuscript, and Mr. Yasuki Tahara and Ms. Mayu Hioki (Nagoya University, Japan) for providing potato tubers.

Funding

This work was supported by the Japan Society for the Promotion of Science under Grant-in-Aid for Scientific Research (B) (26292024) and the Sumitomo Foundation under Grant for Basic Science Research Projects.