Expression analysis of major genes involved in signaling pathways during infection of Chinese cabbage with Hyaloperonospora brassicae

Highlights

• Infection was performed on Chinese cabbage with Hyaloperonospora brassicae.

• Upon infection, only the resistant line exhibited a hypersensitive response.

• Expression changes of genes involved in signaling pathways were compared.

• Resistance in T12–19 is characterized by SA signaling transduction.

• SA signaling pathways in T12–19 followed an R-gene-dependent defense mode.

Abstract

The downy mildew caused by Hyaloperonospora brassicae is a destructive disease for Chinese cabbage (Brassica rapa L. sp. pekinensis), and very few studies have focused on disease resistance mechanisms in this economically important crop. Artificial infection was performed on leaves of resistant and susceptible Chinese cabbage lines with two distinct isolates of H. brassicae. Upon infection with H. brassicae, the resistant line T12–19 initially exhibited a HR. The susceptible line 91–112 did not have hypersensitive response (HR) and showed disease symptoms at initial stages. Plant responses and changes in the expression profiles of genes involved in signaling pathways were compared during the development of infection. Significant differences were found in early transcriptional changes of genes involved in salicylic acid (SA) signaling pathway in line T12–19, but not in the jasmonic acid (JA)/ethylene (ET) signaling pathway. In contrast, early transcriptional changes in the susceptible line 91–112 indicate a weak and abortive basal defense response. Collectively, our results suggest that the mechanism of resistance to downy mildew in resistant Chinese cabbage was in accord with the gene-for-gene model, and the resistance gene activated the SA signaling pathway predominantly to prevent pathogen colonization. However, this does not preclude JA and ET-mediated basal defenses from playing a role in downy mildew resistance.

Introduction

Chinese cabbage (Brassica rapa L. sp. pekinensis) is a major vegetable crop in China that is susceptible to mildew disease caused by Hyaloperonospora brassicae (Göker et al., 2009). During periods of warm temperatures with higher humidity that prevail in spring and fall, this pathogen can cause severe disease epidemics that result in significant crop losses. Most Chinese cabbages are susceptible to the mildew although some differences in severity between lines of resistant and susceptible Chinese cabbage. Fungicides can be effective in controlling infection, but administration of chemicals for organic vegetable production is not feasible. Moreover, improvement of Chinese cabbage for disease resistance through conventional breeding is a cumbersome, long term process. Therefore, effective strategies for controlling the disease are badly needed. In order to develop new control methods, it is necessary to have a thorough understanding of disease resistance mechanisms in Chinese cabbage.

To defend themselves, plants have developed a powerful innate immune system by which they recognize non-self molecules or signals from injured cells, and make accurate defense response (Jones and Dangl, 2006, Howe and Jander, 2008). Defense responses in plants can be classified into two groups based on reports in the literature. One is basal defense, which is not specific to distinct strains of a pathogen and provides partial defense. The other is the gene-for-gene (or race-specific) model of resistance, which requires specific resistance genes (R genes) and provides complete resistance (Feys et al., 2001, McDowell et al., 2001). Plants receive signals, and the innate immune system then activates R genes, resulting in the expression of a series of downstream defense genes encoding antimicrobial proteins or enzymes that catalyze the production of defense metabolites. The major difference between the two forms of resistance is that R gene-dependent defense is accelerated and transient compared to the sustained but delayed induction that occurs in basal defense. However, these two types of resistance share many commonalities, they partially overlap and share components involved in the operation of the response.

Previous studies have reported that the hormones salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are deemed to be the key signaling mediators in plant disease resistance mechanisms (Thomma et al., 1998, Glazebrook, 2001, Grant and Jones, 2009, Pieterse et al., 2009, Leon-Reyes et al., 2010). The SA signaling pathway is classically thought to control resistance to biotrophic pathogens. After detecting microbial pathogens, plants initiate synthesis of SA, and its accumulation subsequently activates NPR1, which plays an important regulatory role in plant defense including the expression of the pathogenesis-related (PR) genes (Cao et al., 1994, Cao et al., 1997, Zhang et al., 1999, Zhou et al., 2000, Pieterse and Van Loon, 2004, Dong et al., 2008). NPR1 monomers are then translocated to the nucleus where they interact with the TGA class of basic leucine zipper transcription factors, leading to the expression of SA-dependent genes (Mou et al., 2003). Recent studies have also implicated WRKY transcription factors in SA defense responses downstream or in parallel with NPR1 (Eulgem and Somssich, 2007). In contrast, JA and ET signaling pathways are responsible for resistance to necrotrophic pathogens (Glazebrook, 2005, Loake and Grant, 2007). In the JA-mediated pathway, defensins (PDF1.2) are considered markers for defense responses. Several studies have demonstrated that the JA signaling pathway includes primary and secondary responses. In the primary response, receptors receive stimulation from the environment, and this results in the activation of several JA biosynthesis genes (Sasaki et al., 2001, Stenzel et al., 2003, Wasternack, 2007). AOS is the key gene in the biosynthesis of JA. In the secondary response, signals are bound to JAR1, which then activates the COI1 gene. COI1 is part of the JA-Ile receptor complex that relays JA-mediated signals via ubiquitylation and degradation of transcriptional repressor JAZ proteins. For ethylene signal transduction, the initial steps involve ETR receptors that localize to the endoplasmic reticulum membrane (Chen et al., 2002, Ma et al., 2006) and receive the signals. As a result, they activate CTR1 which transmits information to downstream signaling components such as EIN2 and the ERF family in the pathway up to nuclear genomic (Meng et al., 2010). Hormone crosstalk is found in SA- and JA/ET-mediated signaling pathways, and antagonism between SA and JA/ET signaling pathways is widely accepted (Adie et al., 2007); however, defense responses are elicited through a complicated network of signaling pathways in plants.

Many studies focused on basis of molecular mechanism and signals for plant disease resistance in important crops, and the development of engineering for plant disease resistance. There have been some reports describing resistance mechanisms against downy mildew on grapevine (Casagrande et al., 2011a, Casagrande et al., 2011b). In resistant grapevines that carry R genes specific for downy mildew, inoculation with two isolates of the pathogen revealed an isolate-specific hypersensitive response (HR). Host reaction relied on transcriptional induction of the HR-associated gene HSR1 and the salicylic acid-induced PR genes PR-1 and PR-2 during the initial 24–48 h post-inoculation, and there were no distinct differences in the expression of genes involved in the JA signaling pathway (Casagrande et al., 2011a, Casagrande et al., 2011b). In contrast, the up-regulation of genes involved in jasmonic acid biosynthesis and the increase in jasmonate levels indicated that this hormone could play a role in resistant species of grapevine against downy mildew (Polesani et al., 2010). Signaling pathways and regulatory elements leading to defense responses in Chinese cabbage have yet to be well understood.

To gain a preliminary understanding of disease resistance mechanisms in Chinese cabbage, we selected 11 genes involved in the SA, JA and ET signaling pathways, and four genes encoding PR proteins, and analyzed transcriptional changes in these genes associated with infection in both susceptible and resistant lines of Chinese cabbage. Our study will provide valuable information toward an understanding of disease resistance mechanisms in Chinese cabbage.