Nicotiana benthamiana MAPK-WRKY pathway confers resistance to a necrotrophic pathogen Botrytis cinerea

ABSTRACT

MEK2-SIPK/WIPK cascade, a Nicotiana benthamiana mitogen-activated protein kinase (MAPK) cascade, is an essential signaling pathway for plant immunity and involved in hypersensitive response (HR) accompanied by cell death. WRKY transcription factors as substrates of SIPK and WIPK have been isolated and implicated in HR cell death. Here, we show virus-induced gene silencing of WRKY genes compromised constitutively active MEK2-triggered cell death in N. benthamiana leaves. In general, HR cell death enhances susceptibility to necrotrophic pathogens such as Botrytis cinerea. However, the WRKY gene silencing elevated susceptibility to B. cinerea. These findings suggest that downstream WRKYs of MEK2-SIPK/WIPK cascade are required for cell death-dependent and -independent immunities in N. benthamiana.

Plants have specific immune systems activated after sensing pathogen by cell surface or intracellular receptors.¹ Mitogen-activated protein kinase (MAPK) cascade is a key signaling pathway of plant defense.² In tobacco, wound-induced protein kinase (WIPK) and salicylic acid-induced protein kinase (SIPK) are known as pathogen-responsive MAPKs^3,4 and share a common upstream MAPK kinase MEK2.⁵ Transient expression of MEK2^DD, a constitutively active MEK2, induces SIPK and WIPK phosphorylation, resulting in defense responses such as hypersensitive response (HR)-like cell death and respiratory burst oxidase homolog (RBOH)-mediated reactive oxygen species (ROS) production in Nicotiana benthamiana leaves.⁶ One of the downstream targets of activated SIPK and WIPK is WRKY transcription factor. N. benthamiana WRKY8, a group I WRKY transcription factor, has in planta phosphorylation motif called SP cluster, and the phospho-mimicking mutant increases its DNA binding and transactivation activities.⁷ In a recent publication, we isolated multiple N. benthamiana WRKYs containing SP cluster as substrates of MAPKs, and WRKY7, 8, 9 and 11 were involved in oomycete effector-triggered ROS production via transactivating machinery of NbRBOHB gene.⁸ Furthermore, Agrobacterium-mediated transient expression of WRKY7, 8, 9 and 11 triggered cell death in N. benthamiana leaves, and the cell death was enhanced by phospho-mimicking mutations in their SP clusters.⁸ These results suggest that WRKY transcription factors can function to induce cell death downstream of the MAPK cascade. This is also supported by the fact that Nicotiana tabacum WRKY1, a substrate of SIPK, contributes to SIPK-triggered cell death.⁹ In this work, to examine involvement of WRKY7, 8, 9 and 11 in MEK2^DD-triggered HR-like cell death, we knocked down the 4 WRKY genes in N. benthamiana by virus-induced gene silencing (VIGS) using WRKY7/8/9/11-silencing construct, which specifically reduces expression levels of the WRKY7/9, 8 and 11 genes by 70%, 68% and 54%, respectively.⁸ VIGS of SIPK and WIPK significantly suppressed the MEK2^DD-triggered cell death, and the cell death was partially compromised or delayed in WRKY7/8/9/11-silenced leaves (Fig. 1A). We measured ion leakage as an index of cell death, and the index showed statistical difference between TRV-control and WRKY7/8/9/11-silenced leaves (Fig. 1B). This finding suggests that WRKY7, 8, 9 and 11 participate in induction of HR-like cell death downstream of MEK2-SIPK/WIPK cascade. In general, triggering cell death at infection sites is a plant defense strategy against biotrophic and hemibiotrophic pathogens, which require living host cells at their infection processes.¹⁰ Our previous observation showed that extension of the infection hyphae of oomycete pathogen Phytophthora infestans, the near-obligate hemibiotroph, was limited by cell death which was compromised by silencing WRKY7, 8, 9 and 11,⁸ suggesting that WRKY7, 8, 9 and 11 redundantly drive the cell death-mediated immunity against P. infestans in N. benthamiana.

Figure 1.

Involvement of WRKYs in MEK2^DD-triggered HR-like cell death. (A) Gene silenced leaves were infiltrated with Agrobacterium strains carrying MEK2^KR or MEK2^DD gene. Photographs were taken 72 h after agroinfiltration. (B) MEK2^DD-triggered cell death was quantified by measuring ion leakage. Asterisks indicate statistically significant differences compared with TRV (t test, **P < 0.01). Data are means ± SD from at least 3 experiments.

Unlike biotrophs, necrotrophic pathogens take advantage of cell death, because they absorb nutrients from dead cells for growth and colonization in their host plants.¹⁰ Botrytis cinerea is a necrotrophic fungi and secretes various extracellular proteins and compounds to degrade host cell wall, generate ROS or induce HR cell death during the infection process.¹¹ One secreted protein BcSpl1, a cerato-platanin family protein known as a virulence factor, elicits HR such as cell death and ROS production in its host tissues.¹² The response partially depends on BRI1-associated kinase 1 (BAK1), a component of pattern recognition receptor complex for sensing pathogens.¹² This observation implies that B. cinerea exploits the elicitor as an infection strategy to activate plant immune system and induce HR symptoms. Our previous study also showed that NbRBOHB-mediated ROS production contributes to expansion of disease lesions by B. cinerea.¹³ WRKY7, 8, 9 and 11 are involved in NbRBOHB-mediated ROS production and cell death induction downstream of MAPK cascade (Fig. 1).⁸ Next, we examined effect of silencing of WRKY7, 8, 9 and 11 genes on disease resistance to B. cinerea in N. benthamiana. WRKY7/8/9/11-silenced leaves were inoculated with B. cinerea by dropping the conidia onto the silenced leaf surfaces. Unexpectedly, WRKY7/8/9/11-silenced leaves showed high susceptibility against B. cinerea compared with TRV control leaves (Fig. 2A). We measured the size of disease lesions to exhibit statistical difference of the susceptibilities (Fig. 2B), indicating that WRKY7, 8, 9 and 11 participate in disease resistance to B. cinerea. In tobacco, loss of function of NtMKP1 gene, coding a MAPK phosphatase targeting SIPK, suppressed expansion of disease lesions by B. cinerea,¹⁴ suggesting that MEK2-SIPK/WIPK cascade positively regulates resistance to B. cinerea. This observation supports our result that WRKYs phosphorylated by SIPK and WIPK are involved in disease resistance to B. cinerea. However, why the cell death-triggering WRKYs are involved in resistance to necrotrophic pathogen? In Arabidopsis, AtWRKY33, the closest WRKY to WRKY8, contains SP cluster phosphorylated by AtMPK3/AtMPK6, orthologs to WIPK and SIPK, respectively, in response to B. cinerea.¹⁵ Moreover, wrky33 mutant shows enhanced susceptibility to B. cinerea.¹⁶ Activated AtWRKY33 by MAPKs targets and up-regulates phytoalexin deficient3 (PAD3) gene in biosynthesis of camalexin, the major Arabidopsis phytoalexin,¹⁵ and pad3 mutant is highly susceptible to B. cinerea.¹⁷ These findings suggest that N. benthamiana MAPK-WRKY pathway could regulate phytoalexin synthesis for disease resistance to necrotrophic pathogen. In fact, we isolated 3-hydroxy-3-methylglutaryl CoA reductase2 (HMGR2) catalyzing biosynthesis of capsidiol, the major tobacco phytoalexin, as a target gene by WRKY8.⁷ In addition, AtWRKY33 was shown as a modulator for balance of plant hormone signaling. Classically, plants are known to use phytohormones for appropriate defense signaling against distinct pathogens, in that salicylic acid (SA) and jasmonic acid (JA) signals appropriately regulate biotrophic and necrotrophic pathogens, respectively.¹⁸ Recent transcriptome analysis using wrky33 mutant upon challenge with B. cinerea indicated that SA-responsive genes were up-regulated in wrky33 mutant, while JA-responsive genes were down-regulated.¹⁹ This implies that WRKYs phosphorylated by MAPKs act for full JA response to B. cinerea. Further studies on downstream signaling of the N. benthamiana WRKYs would provide us a clue to modulating appropriate immune signaling pathways against 2 types of pathogens that have different lifestyles.

Figure 2.

Involvement of WRKYs in resistance to B. cinerea. (A) Gene silenced leaves were inoculated with B. cinerea. Photographs were taken at 3 d postinoculation (dpi). Scale bars, 10 mm. (B) Average diameter of lesions formed on the leaves at 4 dpi. Asterisks indicate statistically significant differences compared with TRV (t test, *P < 0.05).

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank David C. Baulcombe for TRV vector and the Leaf Tobacco Research Center, Japan, for N. benthamiana seeds.

Funding

This work was supported by Grantin-Aid for Scientific Research on Innovative Areas “Oxygen Biology: a new cri-135 terion for integrated understanding of life” (15H01398 to H.Y.) from MEXT of Japan, and by a Grant-in-Aid for Scientific Research (26292023 to H.Y. and 264206 to H.A.) from the Japan Society of the Promotion of Science, by Council for Science, Technology and Innovation (CSTI), Cross-ministerial Strategic Innovation Promotion Program (SIP) of Japan (toH.Y.).