The objective of this study is to clone HSP 9/12 genes of small heat shock proteins without ACD domain from Setosphaeria turcica and analyze their expression patterns during fungal development, infection, and HT-toxin induction processes.

The coding genes of heat shock protein HSP 9/12 were screened and cloned from the whole genome of S. turcica. Bioinformatics methods were employed to analyze the physicochemical properties, subcellular localization, structural prediction, and phylogenetic analysis of HSP 9/12 proteins. RNA-seq and RT-qPCR were used to examine the expression of HSP 9/12 genes during fungal development, infection, and HT-toxin induction.

Two HSP 9/12 genes were screened and cloned from the S. turcica genome, encoding proteins with 99 and 100 amino acids, respectively. Based on their molecular weights, they were named StHsp10.1 and StHsp10.7. Physicochemical analysis revealed that both HSP 9/12 proteins are hydrophilic, with subcellular localization predictions indicating they are located in the cytoplasm with nuclear localization signals. They lack transmembrane domains and signal peptides, and both contain the HSP9_HSP12 (PF04119) domain. StHSP10.1 is an acidic unstable protein, while StHSP10.7 is an alkaline stable protein, both existing predominantly in α-helix-dominated secondary and tertiary structural forms. StHSP10.1 shows closer phylogenetic relationship with Saccharomyces cerevisiae HSP12, whereas StHSP10.7 exhibits closer affinity to Schizosaccharomyces pombe HSP9. The StHSP10.1 exhibited the highest expression during conidial development, followed by hypha, appressoria, and penetration peg, with the lowest expression in germ tubes. After inoculation, the fungal StHSP10.1 expression rapidly increased, reaching 6.37-fold higher FPKM at 72 h compared to 24 h post inoculation. The results of RT-qPCR analysis during the HT-toxin induction process showed that, as the induction time increased, the relative gene expression level of StHSP10.1 in the wild-type strain (WT) significantly increased being 2.9-, 14.1-, and 39.8-fold higher at 14, 21, and 28 d compared to 7 d, respectively, but remained extremely low in the STK1 gene knockout mutant (ΔSTK1). StHSP10.7 showed extremely low expression levels during fungal development, infection, and HT-toxin induction. AlphaFold 3 predicted that the region from -38 to -24 bp upstream of the transcription start site of the StHSP10.1 contains TATA-box, and binding sites for cell differentiation proteins RCD1 and bZIP transcription factor StbZIP11, simultaneously. Using the STRING online platform to construct the protein-protein interaction network for StHSP10.1, two regulatory pathways of StHSP10.1 were proposed: Ras1→STK1→StbZIP11→StHSP10.1 and Ras1→UBE2→CUE1→RCD1-like→StHSP10.1, suggesting important roles in HT-toxin synthesis and stress induction, respectively.

There are significant differences in the expression patterns of HSP 9/12 genes in S. turcica. StHSP10.1 serves as a key regulatory gene in the processes of pathogen development, infection, and HT-toxin induction, whereas StHSP10.7 has no regulatory effect.

The objective of this study is to clone the small heat shock protein (sHSP) genes in Setosphaeria turcica, elucidate their structural characteristics, and explore their expression profiles during pathogen development and in response to HT-toxin induction.

The hidden Markov model (HMM) was used to identify sHSP family members across the entire S. turcica genome. PCR technology was used to clone sHSP genes from S. turcica strain 01-23. Bioinformatics methods were then applied for the analysis of physicochemical properties, subcellular localization, structural prediction, and phylogenetic analysis of the sHSP genes obtained. Additionally, RNA-Seq and RT-qPCR were performed to determine the expression of sHSP genes across different developmental stages and during HT-toxin induction in S. turcica.

Three sHSP family members (StHSP37.2, StHSP37.0 and StHSP22.6) were identified from the genome of S. turcica. The corresponding DNA sequences were successfully cloned from strain 01-23. The encoded sHSP proteins were weakly acidic and hydrophilic proteins, without transmembrane domain or signal peptide. Random coil in the secondary structure accounted for 58.97% to 60.35%, and β-turn ranged from 2.69% to 7.83% only. Subcellular localization prediction indicated that StHSP37.2 and StHSP37.0 were located in the nucleus, while StHSP22.6 was located in both nucleus and cytoplasm. Conserved ACD_sHSP-like domains were identified near C-terminus, with 2, 3, and 5 conserved motifs in StHSP37.2, StHSP37.0, and StHSP22.6, respectively. The monomer tertiary structure models of sHSP were constructed using SWISS-Model and AlphaFill. Phylogenetic analysis indicated close relationships between StHSP22.6 and sHSP in Alternaria alternata, and between StHSP37.2/StHSP37.0 and sHSP in Bipolaris maydis. The sHSP genes of S. turcica had the highest expression levels in hyphae, followed by germ tubes, appressoria, and penetration pegs, with the lowest expression levels in conidia. StHSP22.6 and StHSP37.2 showed significant negative correlations with HT-toxin induction, and the relative gene expression was upregulated by 6.45 and 18.12 folds on day 14, respectively. On day 21 and 28, StHSP37.2 showed modest upregulations of 2.56 and 1.78 folds, respectively, while StHSP22.6 did not differ from the wild-type (WT). StHSP37.0 exhibited significant positive correlations with HT-toxin induction, with a significant downregulation by 59.23%, 86.30%, and 88.11% on day 14, 21, and 28, respectively. Exploration of expressed genes significantly associated with sHSP of S. turcica suggested that StHSP37.2 and StHSP22.6 were mainly related to HSP90, HSP104, catabolism, and mitochondrial Mg²⁺ transport, while StHSP37.0 appeared to be associated with vacuolar alkaline amino acid transport, organic synthesis, and substance secretion.

The sHSP family members in S. turcica demonstrate a high degree of conservation yet exhibit structural and phylogenetic differences from other sHSPs. They are integral to the development of hyphae, germ tubes, appressoria, and penetration pegs, and also exert significant regulatory functions during HT-toxin induction.