Abstract
The haloacid dehalogenase (HAD) superfamily represents a large group of enzymes across diverse taxa. However, the characteristics and functional roles of HAD members in the destructive apple canker pathogen, Valsa mali strain Vm1 (Vm1), remain poorly understood, particularly regarding their expression during infection. In this study, the full-length cDNA sequence of the VmHAD gene from Vm1 was cloned using rapid amplification of cDNA ends (RACE) technology, and its bioinformatic properties, subcellular localization, and expression patterns during infection were characterized. The VmHAD cDNA was 1044 bp in length, containing a complete open reading frame (ORF) of 798 bp that encodes a 265 amino acid protein with a conserved HAD-like domain. Phylogenetic analysis revealed that VmHAD shares the highest similarity with the (S)-2-haloacid dehalogenase (accession no. KUI70710.1) from Cytospora mali 03-8, belonging to the L-2-haloacid dehalogenase family within the HAD hydrolase superfamily. Subcellular localization analysis using a transient expression system in Nicotiana benthamiana indicated that VmHAD is distributed in both the nucleus and cytoplasm. Expression profiling demonstrated that VmHAD was significantly upregulated during the infection of detached apple branches by Vm1, with relative expression levels increasing 3.13-, 4.25-, and 3.98-fold at 3, 5, and 7 days post-inoculation, respectively, compared with day 1, whereas no expression was detected in the uninoculated control. These findings identify VmHAD as a novel HAD family member in Vm1 and suggest that it plays a potential role in the infection process and pathogenicity. This work provides new insights into the molecular mechanisms underlying V. mali pathogenicity and contributes to the development of effective strategies for disease management.