Overexpression of an apple LysM-containing protein gene, MdCERK1–2, confers improved resistance to the pathogenic fungus, Alternaria alternata, in Nicotiana benthamiana

<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Lysin motif (LysM)-containing proteins are involved in the recognition of fungal and bacterial pathogens. However, few studies have reported on their roles in the defense responses of woody plants against pathogens. A previous study reported that the apple <jats:italic>MdCERK1</jats:italic> gene was induced by chitin and <jats:italic>Rhizoctonia solani</jats:italic>, and its protein can bind to chitin. However, its effect on defense responses has not been investigated.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>In this study, a new apple <jats:italic>CERK</jats:italic> gene, designated as <jats:italic>MdCERK1–2</jats:italic>, was identified. It encodes a protein that shares high sequence identity with the previously reported MdCERK1 and AtCERK1. Its chitin binding ability and subcellular location are similar to MdCERK1 and AtCERK1, suggesting that MdCERK1–2 may play a role in apple immune defense responses as a pattern recognition receptor (PRR). <jats:italic>MdCERK1–2</jats:italic> expression in apple was induced by 2 fungal pathogens, <jats:italic>Botryosphaeria dothidea</jats:italic> and <jats:italic>Glomerella cingulate,</jats:italic> but not by the bacterial pathogen, <jats:italic>Erwinia amylovora</jats:italic>, indicating that <jats:italic>MdCERK1–2</jats:italic> is involved in apple anti-fungal defense responses. Further functional analysis by heterologous overexpression (OE) in <jats:italic>Nicotiana benthamiana</jats:italic> (<jats:italic>Nb</jats:italic>) demonstrated that <jats:italic>MdCERK1–2</jats:italic> OE improved <jats:italic>Nb</jats:italic> resistance to the pathogenic fungus, <jats:italic>Alternaria alternata</jats:italic>. H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> accumulation and callose deposition increased after <jats:italic>A. alternata</jats:italic> infection in <jats:italic>MdCERK1–2</jats:italic> OE plants compared to wild type (WT) and empty vector (EV)-transformed plants. The induced expression of <jats:italic>NbPAL4</jats:italic> by <jats:italic>A. alternata</jats:italic> significantly (<jats:italic>p</jats:italic> < 0.01, <jats:italic>n</jats:italic> = 4) increased in <jats:italic>MdCERK1–2</jats:italic> OE plants. Other tested genes, including <jats:italic>NbNPR1</jats:italic>, <jats:italic>NbPR1a</jats:italic>, <jats:italic>NbERF1</jats:italic>, and <jats:italic>NbLOX1</jats:italic>, did not exhibit significant changes after <jats:italic>A. alternata</jats:italic> infection in OE plants compared to EV or WT plants. OE plants also accumulated more polyphenols after <jats:italic>A. alternata</jats:italic> infection.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>Heterologous <jats:italic>MdCERK1–2</jats:italic> OE affects multiple defense responses in <jats:italic>Nb</jats:italic> plants and increased their resistance to fungal pathogens. This result also suggests that <jats:italic>MdCERK1–2</jats:italic> is involved in apple defense responses against pathogenic fungi.</jats:p> </jats:sec>