Identification, Subcellular Localization, and Infection-Related Expression of a Novel Haloacid Dehalogenase Gene (VmHAD) from Valsa mali Vm1

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, <i>Valsa mali</i> strain Vm1 (Vm1), remain poorly understood, particularly regarding their expression during infection. In this study, the full-length cDNA sequence of the <i>VmHAD</i> 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 <i>VmHAD</i> 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 <i>Cytospora mali</i> 03-8, belonging to the L-2-haloacid dehalogenase family within the HAD hydrolase superfamily. Subcellular localization analysis using a transient expression system in <i>Nicotiana benthamiana</i> indicated that VmHAD is distributed in both the nucleus and cytoplasm. Expression profiling demonstrated that <i>VmHAD</i> 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 <i>VmHAD</i> 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 <i>V. mali</i> pathogenicity and contributes to the development of effective strategies for disease management.