LmCBP1, a secreted chitin-binding protein, is required for the pathogenicity of Leptosphaeria maculans on Brassica napus

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Highlights

  • LmCBP1 is a secreted protein and can bind to chitin.

  • Knockout of LmCBP1 in Leptosphaeria maculans using CRISPR-Cas9 lowered the pathogenicity of this pathogen on Brassica napus.

  • The knockout mutants of LmCBP1 were more sensitive to hydrogen peroxide.

Abstract

Leptosphaeria maculans is the causal agent of blackleg disease on Brassica napus. Determining the underlying functions of genes required for pathogenesis is essential for understanding the infection process. A chitin-binding protein (LmCBP1) was discovered as a pathogenicity factor for the infection of B. napus by L. maculans through gene knockout using the CRISPR-Cas9 system. Chitin-binding activity was demonstrated through a chitin-protein binding assay. A secreted signal peptide was detected using a yeast secreted-signal peptide trap assay. An increased expression level during the infection stage was also observed, suggesting that LmCBP1 is a secreted protein. The knockout mutants showed decreased infection on B. napus, with reduced pathogenicity on ten cultivars with/without diverse R genes. The mutants were more sensitive to H2O2 compared to wild type L. maculans isolate JN3. This study provides evidence of the virulence of a novel chitin-binding protein LmCBP1 on B. napus through mutants created via the CRISPR-Cas9 system.

Introduction

Blackleg, caused by Leptosphaeria maculans (L. maculans), is a devastating disease on Brassica napus (B. napus) and leads to enormous economic losses each year in many regions around the world (Fitt et al., 2006). Utilisation of genetic resistance is an effective and environmentally friendly strategy to control the disease. It was suggested that intensive growth of blackleg-resistant canola cultivars with less frequent crop rotation only caused limited yield reductions (Kutcher et al., 2013). There are two layers of resistance against L. maculans in B. napus, including cotyledon resistance controlled by major R genes and adult plant resistance mediated by quantitative resistance genes. In addition, cotyledon resistance is a race-specific resistance and conforms to the gene-for-gene model (Flor, 1971). So far, at least 18 cotyledon R genes and corresponding avirulence genes (Avr) have been identified (Ansan-Melayah et al., 1998, Balesdent et al., 2002, Balesdent et al., 2005, Balesdent et al., 2013, Chevre et al., 1996, Delourme et al., 2004, Keri et al., 1997, Larkan et al., 2013, Parkin et al., 1995, Van de Wouw et al., 2009, Yu et al., 2005, Yu et al., 2007, Yu et al., 2013), and nine of these Avr genes have been cloned (Balesdent et al., 2013, Fudal et al., 2007, Ghanbarnia et al., 2015, Gout et al., 2006, Parlange et al., 2009, Plissonneau et al., 2016, Plissonneau et al., 2017b, Van de Wouw et al., 2014). All of these cloned Avr genes were predicted to encode small secreted proteins (SSPs) with secreted signal peptides, and one of them, AvrLm1 was validated as a secreted protein (Ma et al., 2018). AvrLm3 was shown to be a pathogenicity factor in attacking B. napus (Plissonneau et al., 2017a). Therefore, discovering and clarifying the function of these SSPs should be crucial for understanding the underlying pathogenicity of L. maculans.

As a major component of fungal cell walls, chitin acts a pathogen-associated molecular pattern (PAMP) in plants and animals (Boller, 1995, Felix et al., 1993, Lee et al., 2008). It can be targeted by the host plant chitinases (Mauch et al., 1988, Jongedijk et al., 1995, Sela-Buurlage et al., 1993) and by the LysM receptors (Xue et al., 2019), which can trigger plant defence responses through the MAPK cascade and transcription factor network (Kaku et al., 2006, Miya et al., 2007, Wan et al., 2008a, Wan et al., 2008b). However, pathogens can evolve to produce effectors for overcoming PAMP-triggered immunity (PTI) (Cunnac et al., 2009). Some of these avirulence effectors can overcome the PTI through suppressing PTI directly, such as the avirulence effector AvrPiz-t of Magnaporthe oryzae, Phytophthora sojae effector Avr1b and Fusarium oxysporum f.sp. lycopersic effector Avr1. (Dou et al., 2008, Houterman et al., 2008, Park et al., 2012). Another way that an avirulence effector overcomes the PTI is by strengthening pathogen cells to protect pathogens from the PTI.

Avr4, an effector, can specifically bind to the chitin in fungal cell walls to protect fungal hyphae against hydrolysis by plant chitinases (van den Burg et al., 2006, Westerink et al., 2002). Another example is the Parastagonospora nodorum effector, SnTox1, which can protect different pathogens from chitinases (Liu et al., 2016). Though L. maculans is one of the most important pathogens of B. napus, there are no reports on the critical role of chitin-binding proteins in L. maculans. In this study, we used the CRISPR-Cas9 system to clarify the function of a chitin binding protein, LmCBP1.

In this study, LmCBP1 was identified as a secreted chitin binding protein but did not contain any chitinase or cellulase activity. Knockout mutants showed reduced susceptibility on different B. napus cultivars, and the reduced susceptibility was not affected by the absence/presence of R genes in those cultivars. Levels of cell death were reduced in cotyledons of B. napus cultivars inoculated with lmcbp1 mutants compared with the wild type isolate. This research indicates that the fungal pathogen L. maculans utilises secreted proteins, to protect itself from the toxicity of hydrogen peroxide (H2O2) and affect the resistance of the host to aid its colonisation, providing important insights into the molecular basis of the B. napus-L. maculans interaction.

Section snippets

Fungal and plant materials

The L. maculans wild-type strain JN3 was used in this study, and it contained seven avirulence genes: AvrLm1, AvrLm4, AvrLm5, AvrLm6, AvrLm7, AvrLm8 and AvrLepR1. The wild-type and transformed isolates were maintained on 10% V8 agar (1 L agar medium consists of 200 mL V8 juice, 0.75 g CaCO3, and 15 g agar) in Petri dishes. B. napus cultivars MT29 (Rlm1, 9), Glacier (Rlm 2, 3), 01–22-2–1 (Rlm3), Jet Neuf (Rlm4), Forge (Rlm6), 01-23-2 (Rlm7), Goéland (Rlm9), 1135 (LepR2), Surpass400 (LepR3, RlmS)

LmCBP1 is a chitin binding protein without chitinase or cellulase activity

From previous research, we found that the gene Lema_T124370.1 had a higher expression level during the infection process of L. maculans on B. napus cotyledons (Haddadi et al., 2016). Sequence analysis indicated that the protein encoded by Lema_T124370.1 (belongs to Auxiliary Activity Family 16) contained a signal peptide and a LPMO_10 domain (Fig. 1A). The LPMO_10 domain can be found in a variety of cellulose- and chitin-binding proteins (Forsberg et al., 2014). The phylogenetic analysis

Discussion

In this research, we characterised a chitin-binding protein called LmCBP1 in L. maculans. The first 19 amino acids of LmCBP1 were predicted by the signal peptide prediction program SignalP5.0 (Armenteros et al., 2019). We then validated the 19 amino acids as a typical signal peptide using the yeast signal sequence trap method (Yin et al., 2018). Six cysteine residues, as well as the formation of disulphide bonds, were predicted using the web-based program DISULFIND (Ceroni et al., 2006). The

Acknowledgments

We greatly appreciate the efforts of Paula Parks for her thorough reading and editing of this manuscript. This research was supported by the SaskCanola and NSERC-CRD and NSERC Discovery grants awarded to W.G.D. Fernando.

Declaration of Competing Interest

All authors have approved and there is no conflict of interest for the revised manuscript.

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