ERECTA contributes to non-host resistance to Magnaporthe oryzae in Arabidopsis

Abstract

ERECTA controls both developmental processes and disease resistance in Arabidopsis. We investigated the function of ERECTA in non-host resistance to Magnaporthe oryzae in Arabidopsis. In the pen2 er mutant, penetration resistance and post-penetration resistance to M. oryzae were compromised. These results suggest that ERECTA is involved in both penetration and post-penetration resistance to M. oryzae in Arabidopsis.

Key words:

• Arabidopsis
• ERECTA
• Magnaporthe oryzae
• non-host resistance

Plants have evolved effective basal defense systems to detect and limit the growth of pathogens. Pathogens can be recognized by the host through the perception of conserved pathogen-associated molecular patterns (PAMPs). PAMPs are recognized by transmembrane pattern recognition receptors (PRRs) that bind with specific PAMPs and initiate intracellular immune responses. Plant PRRs are either surface-localized receptor-like kinases or receptor-like proteins that contain various ligand-binding ectodomains that perceive PAMPs or damage-associated molecular patterns (DAMPs).¹⁾

Disease resistance shown by an entire plant species to all genetic variants of a non-adapted pathogen species is the most common form of plant immunity and termed non-host resistance (NHR).²⁾ Arabidopsis mutants with altered non-host interactions following non-adapted powdery mildew Blumeria graminis hordei infection have been described, and three genes have been identified: PENETRATION 1 (PEN1), PEN2, and PEN3.^3–6) PEN1 encodes a plasma membrane-anchored syntaxin with a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) domain.³⁾ PEN2 encodes an atypical myrosinase involved in glucosinolate metabolism in defense responses.^4,7,8) PEN3 encodes a pleiotropic drug resistance-type (PDR) ATP-binding cassette transporter.^5,6) Although the identified pen mutants (pen1, pen2, and pen3) compromise NHR at the cell periphery, each mutant line retains the ability to mount effective post-invasion immunity, which is linked to a cell death response. Collectively, these studies demonstrated that Arabidopsis NHR to non-adapted biotrophic powdery mildews has two successive multicomponent defense layers: penetration resistance and post-penetration resistance.

Rice blast caused by Magnaporthe oryzae is a devastating disease of rice. The mechanisms of resistance to M. oryzae have been extensively studied, and the rice-M. oryzae pathosystem has become a model system in plant–microbe interaction studies.^9,10) However, the mechanisms of NHR to M. oryzae in other plants remain poorly understood. We have found that PEN2, powdery mildew resistance 5 (PMR5), Arabidopsis G-protein beta subunit 1 (AGB1), and mildew locus O 2 (MLO2) are involved in both penetration resistance and post-penetration resistance to M. oryzae in Arabidopsis.^11–16) We have also found that penetration resistance to M. oryzae in Col-0 was stronger than that of Ler in Arabidopsis. To examine the genetic basis underlying natural variation in the responses, we used a set of recombinant inbred lines derived from a Col × Ler cross and identified three quantitative trait loci (QTL) that govern the expression of NHR in Arabidopsis against M. oryzae. Among the three QTL, QTL2 on chromosome 2 contained an ERECTA coding sequence.¹¹⁾ The leucine-rich repeat receptor-like kinase ERECTA controls both developmental processes and disease resistance in Arabidopsis.¹⁷⁾ ERECTA has been identified as a positive mediator of resistance against bacteria (Ralstonia solanacearum),¹⁸⁾ fungi (Plectosphaerella cucumerina and Verticillium longisporum),^19,20) and oomycetes (Pythium irregulare).²¹⁾ The deposition of β-1,3-glucan callose, a defense response of plants to pathogen attack or PAMP treatment, was impaired in the er mutant upon Pl. cucumerina inoculation, but not after inoculation with Pl. parasitica (Peronospora parasitica) or treatment with the flg22 elicitor.¹⁹⁾

In this study, we examined the function of ERECTA in NHR to M. oryzae in Arabidopsis.

Arabidopsis plants were grown under short-day conditions (9:15 L:D) at 22 °C in a growth room. The Arabidopsis accession code was Col-0. We used the following mutants: pen2–1⁴⁾ and er (SALK_044110)^22,23) (all with the Col-0 background). The pen2 er double mutant was generated by crossing er with pen2–1. Plants homozygous for both er and pen2–1 loci were identified in the F2 progeny by PCR genotyping, as previously described.^11,13) Fungal inoculation and quantification of cell entry and fungal growth were conducted as previously described.^11–16) Data were compared using Tukey’s highly significant difference (HSD) tests. Calculations were performed on three data-sets (n = 3), and p < 0.05 indicated statistically significant effects. For visualization of callose, samples were stained with aniline blue as previously described.¹¹⁾

To determine whether ERECTA would affect NHR to M. oryzae in Arabidopsis, the er mutant was inoculated with M. oryzae and monitored by microscopy. Analysis of the M. oryzae challenge for the er mutant at 72-h post-inoculation (hpi) revealed that the rate of entry was slightly higher than that of the wild-type plants, although there were no significant differences between them (Fig. 1). Double mutants were generated between the pen2 and er mutants to evaluate the role of ERECTA in NHR to M. oryzae in plants with a pen2 background. We harvested leaves of infected plants at 72 hpi and examined them microscopically. The rate of entry into pen2 er plants was significantly higher than that into pen2 plants (Fig. 1).

Fig. 1. Quantitative analysis of penetration resistance to M. oryzae in Arabidopsis mutants.

Notes: Mean frequency of M. oryzae penetration into Arabidopsis mutants at 72-h post-inoculation (hpi) expressed as percentage of the total number of infection sites. Values are means ± standard errors, n = 3 independent experiments. Bars sharing the same lowercase letters are not significantly different (p > 0.05). col, Col-0; p2, pen2; p2 er, pen2 er.

We investigated the role of ERECTA in post-penetration resistance by measuring the lengths of the longest infection hyphae in pen2 er plants at 72 hpi. The infection hyphae in pen2 er plants were significantly longer than those in pen2 plants (Fig. 2(A)). We also examined the proportion of branched hyphae development in pen2 er plants. The proportion of branched hyphae development in pen2 er plants was slightly higher than that in pen2 plants, although there were no significant differences between them (Fig. 2(B)).

Fig. 2. Quantitative analysis of post-penetration resistance to M. oryzae in Arabidopsis mutants.

Notes: (A) Mean length of infection hyphae was measured at 72 hpi. Values are means ± standard errors (n = 6) from three biological replicates. Bars sharing the same lowercase letters are not significantly different (p > 0.05); (B) Mean frequency of M. oryzae penetration with branched hyphae into Arabidopsis mutants at 72 hpi expressed as a percentage of penetrated cells. Values are means ± standard errors, n = 3 independent experiments. Bars sharing the same lowercase letters are not significantly different (p > 0.05). p2, pen2; p2 er, pen2 er.

To determine whether ERECTA would affect callose accumulation in NHR to M. oryzae in Arabidopsis, infection sites were stained with aniline blue. We harvested leaves of the infected plants at 72 hpi and examined them microscopically. UV-induced fluorescence with aniline blue was detected in invaded epidermal cells. We found that callose accumulated in pen2 er plants after M. oryzae infection (Fig. 3).

Fig. 3. Accumulation of callose at infection sites in Arabidopsis mutants.

Notes: (A) Light microscopic view of infection sites of pen2 plants at 72 hpi; (B) Callose deposition at infection sites in (A) as visualized by fluorescence microscopy following aniline blue staining; (C) Light microscopic view of infection sites of pen2 er plants at 72 hpi; (D) Callose deposition at infection sites in (C) as visualized by fluorescence microscopy following aniline blue staining. a, Appressorium. Bars = 50 µm.

In conclusion, penetration resistance and post-penetration resistance in the pen2 er mutant were compromised. This suggests that ERECTA is a positive regulator of NHR to M. oryzae in Arabidopsis.

Author contributions

A.I. designed the research; T.T., H.S., and A.I. performed the research; A. I. wrote the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by JSPS KAKENHI [grant number 26450058].

Acknowledgments

We would like to acknowledge ABRC for providing seeds of Col-0 and er (SALK_044110). We thank Dr H. Koga (Ishikawa Prefectural University) for providing the M. oryzae isolate and Dr P. Shulze-Lefert (Max Planck Institute for Plant Breeding Research) for seeds of pen2-1.