## 2 Results  
Identification, Phylogeny, and Transcriptomic Dynamics of Genes Coding for TBCC Domain-Containing Proteins in P. capsaicin  
The nine tubulin-binding cofactor C domain-containing proteins in _P. capsaicin_ were identified by performing BLASTp and reverse-BLASTp search analyses with amino acid sequences of TBC domain-containing proteins retrieved from the Ensembl Genomes ([[http://ensemblgenomes.org/](http://ensemblgenomes.org/)]([http://ensemblgenomes.org/](http://ensemblgenomes.org/)), accessed on 09 February 2022) database [14] for _Schizosaccharomyces pombe_, _Neurospora crassa_, _Aspergillus nidulans_, _Magnaporthe oryzae_, _Chlamydomonas reinhardtii_, _Styela clava_, _Stegodyphus dumicola_, _Breviolum minutum_, _Capsicum annuum_, _Volvox carteri f. nagariensis_, _Arabidopsis thaliana_, _Aureococcus anophagefferens_, _Saprogenia parasitic_,_Phytophthora sojae_, and _Phytophthora infestans_. Further phylogenetic examinations showed that PcTbcc shared evolutionary ties with _P. infestans_, _S. parasitica_, and _A. anophagefferens_ but was evolutionarily distant from _P. sojae_ (Figure 1a). Protein sequence feature analysis showed that, in addition to the conserved TBCC domain identified in this study, protein sequences from plants and fungi possess an additional TBCC_N domain. Interestingly, we also showed that while SpTbcc and AaTbcc possess nuclear-targeting peptides (nTPs), PcTbcc1 and SpaTbcc contain an exclusive chloroplast-targeting peptide (cTP) and mitochond-targeting peptide (mTP), respectively (Figure 1b). These results indicate a possible divergence in the functions of TBCC domain-containing proteins in oomycetes compared to plants and fungi. We monitored the expression patterns of nine _P. capsici_ TBC domain-containing coding genes during the early and late stages of pathogen-host interactions. Comparative transcriptomic profiling revealed a significant upregulation of TBCA (DVH05_026725), TBCC1 (DVH05_025197), TBCC-2 (DVH05_019376), and TBCE (DVH05_001436), with TBCC1 exhibiting the highest expression intensities in the early and late stages of _P. capsici_ interactions with pepper, tomato, squash, faba bean, kidney bean, and watermelon seedlings (Figure 1c). Based on these observations, we hypothesized that TBCC1 functions as a crucial pathogenic determinant that regulates the pathogenicity and virulence of _P. capsici_ in multiple hosts. Figure 1: Phylogenetic relationship, domain architecture, and expression pattern of _PcTBCC1_ during _P. capsici_–pepper interaction. (**a**) The cladogram shows the prevailing phylogenetic relationship between TBCC domain-containing proteins from selected organisms. MEGA X was used to construct the neighbor-joining trees. (**b**) The trees represent the comparative structure and distribution of the TBCC domain in TBCC domain-containing proteins identified in the selected organisms. (**c**) The heatmap represents the expression of the individual genes coding for TBCC domain-containing proteins identified in _P. capsici_ at early (8–24 hpi) and late (36–72 hpi) stages of pathogen–host interaction. The RNA sequencing data were obtained using a deep-sequencing approach, and the individual expression patterns were consistent in all seven replicates. The gene expression level was computed using the RFPKM method. * highlight the position of PcTbcc in the phylogenetic tree and on the Heatmap. PcTbcc1 Functions as a Negative Genetic Regulator of Vegetative Growth in P. capsaicin Under Nutrient-Deficient Conditions  
We examined the likely effects of PcTbcc1 dysfunctions on the vegetative development of _P. capsici_ by comparing the colony diameters of the _TBCC1_ targeted gene deletion strains (\(\Delta\)_PcTbcc1-212_ and \(\Delta\)_PcTbcc1-297_, TBCC domain-defective strains (_PcTbcc1-72\(\Delta\)_115-611_ and _PcTbcc1-124\(\Delta\)_115-611_), the strains harboring the empty pYF515 and pBS plasmids (EV), and the wild-type strains cultured on clarified V8 juice agar medium.