Characterization of the Largest Secretory Protein Family, Ricin B Lectin-like Protein, in Nosema bombycis: Insights into Microsporidian Adaptation to Host

Microsporidia are a group of obligate intracellular pathogens infecting nearly all animal phyla. The microsporidian Nosema bombycis has been isolated from several lepidopteran species, including the economy-important silkworms as well as several crop pests. Proteins secreted by parasites can be important virulent factors in modulating host pathways. Ricin is a two-chain lectin best known for its extreme vertebrate toxicity. Ricin B lectin-like proteins are widely distributed in microsporidia, especially in N. bombycis. In this study, we identify 52 Ricin B lectin-like proteins (RBLs) in N. bombycis. We show that the N. bombycis RBLs (NbRBLs) are classified into four subfamilies. The subfamily 1 was the most conserved, with all members having a Ricin B lectin domain and most members containing a signal peptide. The other three subfamilies were less conserved, and even lost the Ricin B lectin domain, suggesting that NbRBLs might be a multi-functional family. Our study here indicated that the NbRBL family had evolved by producing tandem duplications firstly and then expanded by segmental duplications, resulting in concentrated localizations mainly in three genomic regions. Moreover, based on RNA-seq data, we found that several Nbrbls were highly expressed during infection. Further, the results show that the NbRBL28 was secreted into host nucleus, where it promotes the expressions of genes involved in cell cycle progression. In summary, the great copy number, high divergence, and concentrated genome distribution of the NbRBLs demonstrated that these proteins might be adaptively evolved and played a vital role in the multi-host N. bombycis.


Introduction
Microsporidia are a group of obligate intracellular parasites, which can infect a wide variety of hosts from protists to mammals, even humans [1][2][3][4]. Nosema bombycis, a kind of parasite of the silkworm (Bombyx mori), was the first formally described microsporidia and has been shown to be transmitted both vertically and horizontally. Infection of N. bombycis will result in the death of its host, thus posing a significant threat to sericulture industry [5]. As intracellular parasites, microsporidia utilize many of its host metabolites to reduce genomes and thus speed up reproduction [6,7].
Ricin, notable for its extreme vertebrate toxicity, is a heterodimeric protein (carbohydrate containing protein) and is found in the seeds of the castor oil plant Ricinus communis L [8]. It has a cell-binding ricin toxic B chain (RTB) linked, through a disulfide bound, to a catalytic cytotoxic ribosome-inactivating protein (RTA). The RTB has two sugar-binding regions, each of which contain three homologous subregions (alpha, beta, and gamma) composed of
Conserved motifs of the SP were identified using MEME 5.3.0 [23] with default parameters, except for the maximum number of motifs and maximum width, which were set to 4 and 10, respectively. Only motifs with an e value < 1e −22 were kept for further analysis.

Expressions of Nbrbls during N. bombycis Infection
N. bombycis spores were prepared as previously described [27]. The spores, which were pretreated with 0.1 mol/L KOH, were added to the BmE-SWU1 cells (cell:spore ratio, 1:20). Infected cells were collected at 12-, 24-, 48-, 60-and 72-h post infection (hpi) and stored in TRIzol (Ambion, CA, USA). RNA extraction and cDNA synthesis were performed as previously described [28]. The real-time quantitative PCR (RT-qPCR) was conducted using primers (Nbrbl16, Nbrbl45, and Nbrbl51) and reference gene Nbtubulin (Table 1). Expression levels were calculated by the 2 −∆∆t values method using three replicates. All statistical t-tests were performed with GraphPad Prism version 9.0.0 by two-tailed comparison tests and any difference with a p-value < 0.05 was considered significant [29].
TTCGTACTGGCTCTTCTCGT CAAAGTTGATAGCAATTCCCT Transcriptomic data were downloaded from scientific publications (the accession number PRJNA549766) as reference and were used to analyze the expression patterns of NbRBL proteins [30].

Indirect Immunofluorescence Assay (IFA)
Infected BmE-SWU1 cells were fixed with 4% paraformaldehyde for 10 min at room temperature and washed three times with 1xPBS and permeabilized using 0.1% Triton X-100 for 15 min. The cells were then blocked in 1xPBST containing 5% BSA and 10% goat serum for 1 h at room temperature. Next, the cells were incubated with mouse and rabbit poly clonal antibodies against NbRBL06 (anti-NbRBL06) and NbRBL28 (anti-NbRBL28) diluted 1:100 in blocking solution for 2 h at room temperature. The cells were then washed for three times with 1xPBST, and incubated for 1 h with a 1:1000 dilution of Alexa Fluor 488 conjugate Goat anti-Mouse IgG (Invitrogen A32723, Rockford, Illinois, USA) and Alexa Fluor 594 conjugate Goat anti-Mouse IgG (Invitrogen A32742, Rockford, Illinois, USA) in a dark moist chamber at room temperature. The cell nucleus was stained with DAPI (1:1000 dilution, Sigma-Aldrich 28718-90-3, St. Louis, MO, USA) at room temperature for 15 min. The samples were finally observed and photographed using an Olympus FV1200 laser scanning confocal microscope.

Transfection and RNA-seq
The Nbrbl28 (locus NBO_163g0001) was cloned from N. bombycis genomic DNA and inserted into the pSL1180 over expression vector fused with egfp. BmE-SWU1 cells were transfected with Nbrbl28::egfpand egfp-containing plasmids according to the instructions of X-tremeGENE HP DNA transfection reagents (Roche 06366546001). The cell culture medium was replaced with fresh Grace's Insect medium containing 10% serum after 5 h. Three days later, the transfected cells were collected, and stored at −80 • C for RNA-seq. RNA-seq was conducted by the Biomarker Technology Company (Beijing, China). The raw data were deposited in GenBank under the BioProject PRJNA808047 and BioSample SAMN26022686. The real-time quantitative PCR (RT-qPCR) was conducted using primers E2F1, SDS3, and Rad51 and reference gene primer SW22934. The reaction procedure included one cycle at 95 • C for 5 min, followed by 40 cycles at 95 • C for 10 s and at 60 • C for 30 s. Expression levels were calculated by the 2 −∆∆t values method using three replicates.

The Nbrbls Identified in N. bombycis Genome
A total of 52 Nbrbls were identified in the N. bombycis genome, composing the largest protein family in N. bombycis. As shown in Table 2, the pI of different NbRBLs was variable, ranging from 4.55 to 9.13. The molecular weight of the NbRBLs is from 20 to 35 kDa. Thirty of 52 NbRBLs contain a Ricin B lectin domain (RBLD). The deficiency of RBLD was also found in other microsporidia [14].

The NbRBLs Are High Divergent
Phylogeny analysis shows a high level of divergence among the NbRBLs, which can be grouped into 4 subfamilies containing 17, 25, 7, and 3 members ( Figure 1). Subfamily 1 is relatively conserved in that all members contain the RBLD, 14 of which encode a signal peptide (SP). Some members of the other subfamilies have lost RBLD, suggesting that these members are more differentiated. In subfamily 2, 17 out of 25 members have lost the RBLD, and only half retain the SP. Again, in subfamily 3, some members show a loss of the SP, RBLD. All members of subfamily 4 have the SP, while only one has the RBLD. Furthermore, there were 9 members with a SP and a nuclear localization signal (NLS), indicating that these factors could be secreted into host nucleus. In summary, the NbRBLs are a highly differentiated protein family that may have diverse functions in parasites.

Expansive Mechanisms of the NbRBL Family
By mapping N. bombycis genome [17], we found that the 52 Nbrbls are located on 20 scaffolds (Figure 2a). Tandem gene duplications were found on scaffolds NBO_6, NBO_27, NBO_463, and NBO_1196, containing 19, 6, 5, and 4 genes, respectively. Moreover, we found that the members of each NbRBL subfamily were distributed on the differ- Phylogenetic analysis of NbRBLs. The phylogenetic tree was constructed using RaxML [24] with the Maximum Likelihood model from multiple sequence alignment of NbRBLs and visualized using the iTOL (http://itol.embl.de/, accessed on 15 September 2021). The NbRBL family was divided into four subfamilies. Branches in same background color indicate members in a subfamily. RICOM RBL, the castor ricin B chain (GenBank accession no, ACY38598.1), was used as the out group.

Expansive Mechanisms of the NbRBL Family
By mapping N. bombycis genome [17], we found that the 52 Nbrbls are located on 20 scaffolds (Figure 2a). Tandem gene duplications were found on scaffolds NBO_6, NBO_27, NBO_463, and NBO_1196, containing 19, 6, 5, and 4 genes, respectively. Moreover, we found that the members of each NbRBL subfamily were distributed on the different scaffolds. Members of each NbRBL subfamily were distributed on the NBO_6, but on the NBO_463, there only existed members of subfamily 1, and on the NBO_27 and NBO_1196 Scaffold, there were only members of subfamily 2. The NbRBL family formed clusters in the N. bombycis genome, indicating that the Nbrbls experienced large-scale duplication. In the largest region containing the tandem duplications (TDs) and segmental duplications (SDs) of Nbrbl, we found transposable elements (TEs) flanking the SD region (Figure 2b).

The Reduction of Key Motifs in NbRBL
The Ricin B lectin domain has been referred to as the (QxW)3 domain and the three homologous regions as the QxW repeats. Through multiple sequence alignment of NbRBL family, it is found that family 1 is relatively conservative in all subfamilies. Compared with RTB of castor, subfamily 1 also has three distinct subdomains: α, β, and γ. However, there is no obvious QxW motif in the α subdomain, even the QxW motif in α subdomains turns into QxF motif, which is found in three other three families (Figures 3 and S1-S3).

The Reduction of Key Motifs in NbRBL
The Ricin B lectin domain has been referred to as the (QxW) 3 domain and the three homologous regions as the QxW repeats. Through multiple sequence alignment of NbRBL family, it is found that family 1 is relatively conservative in all subfamilies. Compared with RTB of castor, subfamily 1 also has three distinct subdomains: α, β, and γ. However, there is no obvious QxW motif in the α subdomain, even the QxW motif in α subdomains turns into QxF motif, which is found in three other three families (Figures 3 and S1-S3). We also analyzed the sequence feature of the SPs in NbRBL and found that thei lengths ranged from 12 to 24 (Figure 4). In addition, some researchers have found that there is a conserved amino acid sequence PEXEL/VTS/HT at the N-terminus of most secreted proteins of Plasmodium, which enable secreted proteins to pass through the vacuole [1,31] We found a conserved amino acid sequence in the SP of NbRBL family members. It i speculated that they have similar secretory pathway and are secreted into host cells to play a regulatory role. (a) NbRBL subfamily 1 has three distinct subdomains: α, β, and γ, which have same structure as the RTB of castor. However, there is no obvious QxW motif in α subdomain, and the QxW motif in α subdomains turns into QxF motif (the green box). (b) Amino acid molecular structure of tryptophan and phenylalanine. Compared with tryptophan, the molecular weight of phenylalanine is smaller, and the structure becomes simpler.
We also analyzed the sequence feature of the SPs in NbRBL and found that their lengths ranged from 12 to 24 (Figure 4). In addition, some researchers have found that there is a conserved amino acid sequence PEXEL/VTS/HT at the N-terminus of most secreted proteins of Plasmodium, which enable secreted proteins to pass through the vacuole [1,31]. We found a conserved amino acid sequence in the SP of NbRBL family members. It is speculated that they have similar secretory pathway and are secreted into host cells to play a regulatory role.

Expressions of Nbrbls during Infection
N. bombycis can be transmitted vertically from infected females to eggs, resulting in congenital infections in embryos. Based on the RNA-seq data from articles published in the scientific literature [30], we analyzed the expression patterns of Nbrbls in B. mori embryos infected with N. bombycis and found that 26 of the identified Nbrbls were expressed during infection in N. bombycis. No expression of NbRBL subfamily 4 was detectable, Members of all other subfamilies showed expression during infection. Among them, 14 of 17 members in subfamily 1 were expressed. In addition, we found that three NbRBLs from subfamily 2 (Nbrbl51) and subfamily 3 (Nbrbl16 and Nbrbl45) were highly expressed. Apart from Nbrbl06, most of the members of subfamily 1 showed a lower level of expression. Five genes from subfamily 1 (Nbrbl05, Nbrbl08, Nbrbl09 Nbrbl17, and Nbrbl18) were highly expressed early on and down-regulated during embryos development ( Figure 5). We further analyzed the expression patterns of Nbrbls in the infected BmE-SWU1 cells. The results showed that the overwhelming majority of Nbrbls were expressed and Nbrbl16, Nbrbl45, and Nbrbl51 were highly expressed (Figure 6a). Then, we selected three genes (Nbrbl16, Nbrbl45, and Nbrbl51), highly expressed to examine their expression profile in the BmE-SWU1 cells after N. bombycis infection (Figure 6b). The data showed that the expression of these three genes were up-regulated at 48 hpi. Figure 3. Multiple sequence alignment of NbRBL subfamily 1. (a) NbRBL subfamily 1 has three distinct subdomains: α, β, and γ, which have same structure as the RTB of castor. However, there is no obvious QxW motif in α subdomain, and the QxW motif in α subdomains turns into QxF motif (the green box). (b) Amino acid molecular structure of tryptophan and phenylalanine. Compared with tryptophan, the molecular weight of phenylalanine is smaller, and the structure becomes simpler.
We also analyzed the sequence feature of the SPs in NbRBL and found that their lengths ranged from 12 to 24

amino acids. A conserved amino acid motif [ILF][LI][LIF][IV][LFI][SK][IL]IK[ASC]
was predicted, demonstrating that NbRBLs have similar secretion pathways (Figure 4). In addition, some researchers have found that there is a conserved amino acid sequence PEXEL/VTS/HT at the N-terminus of most secreted proteins of Plasmodium, which enable secreted proteins to pass through the vacuole [1,31]. We found a conserved amino acid sequence in the SP of NbRBL family members. It is speculated that they have similar secretory pathway and are secreted into host cells to play a regulatory role.

Expressions of Nbrbls during Infection
N. bombycis can be transmitted vertically from infected females to eggs, resulting in congenital infections in embryos. Based on the RNA-seq data from articles published in the scientific literature [30], we analyzed the expression patterns of Nbrbls in B. mori embryos infected with N. bombycis and found that 26 of the identified Nbrbls were expressed during infection in N. bombycis. No expression of NbRBL subfamily 4 was detectable, Members of all other subfamilies showed expression during infection. Among them, 14 of 17 members in subfamily 1 were expressed. In addition, we found that three NbRBLs from subfamily 2 (Nbrbl51) and subfamily 3 (Nbrbl16 and Nbrbl45) were highly expressed. Apart from Nbrbl06, most of the members of subfamily 1 showed a lower level of expression. Five genes from subfamily 1 (Nbrbl05, Nbrbl08, Nbrbl09 Nbrbl17, and Nbrbl18) were highly expressed early on and down-regulated during embryos development ( Figure 5). We further analyzed the expression patterns of Nbrbls in the infected BmE-SWU1 cells. The results showed that the overwhelming majority of Nbrbls were expressed and Nbrbl16, Nbrbl45, and Nbrbl51 were highly expressed (Figure 6a). Then, we selected three genes (Nbrbl16, Nbrbl45, and Nbrbl51), highly expressed to examine their expression profile in the BmE-SWU1 cells after N. bombycis infection (Figure 6b). The data showed that the expression of these three genes were up-regulated at 48 hpi.  [30]. Each column represents a time-point, each row represents a gene. For detailed FPKM of Nbrbls, see Table S1. bombycis-infected embryo of B. mori after 1-9 days post oviposition (dpo) was calculated from the RNA-Seq data we published before [30]. Each column represents a time-point, each row represents a gene. For detailed FPKM of Nbrbls, see Table S1.

Subcellular Localization of NbRBL16 and NbRBL28
First, we verified the specificity of the antibody using Western blotting, which revealed that the antibody of NbRBL16 and NbRBL28 distinguishes these endogenous proteins from the total proteins in N. bombycis infected cell (Figure 7a). The NbRBL16 protein is located in the cytoplasm of schizont in the proliferating stage while the mature spores gave no fluorescent signal (Figure 7b), similar to that of Cyto-NbHsp70 [28]. NbRBL28 contains an N-terminal signal peptide and nuclear localization signal (NLS) sequences (Figure 1), which was co-expressed with EGFP in BmE-SWU1 to assess whether it could be secreted into the host nucleus. As expected, we found that four NbRBL28 proteins could be located in the BmE-SWU1 cell nucleus (Figure 7c). Although NbRBL28-EGFP fusion protein was located in the host nucleus, NbRBL28 could be secreted into the host nucleus during N. bombycis infection. The result showed that NbRBL28 was not only located in the cytoplasm of the schizont, but was also detected in the host cell nucleus (Figure 7d), which demonstrated that NbRBL28 was a secreted protein targeted to the host cell nucleus.

Subcellular Localization of NbRBL16 and NbRBL28
First, we verified the specificity of the antibody using Western blotting, which revealed that the antibody of NbRBL16 and NbRBL28 distinguishes these endogenous proteins from the total proteins in N. bombycis infected cell (Figure 7a). The NbRBL16 protein is located in the cytoplasm of schizont in the proliferating stage while the mature spores gave no fluorescent signal (Figure 7b), similar to that of Cyto-NbHsp70 [28]. NbRBL28 contains an N-terminal signal peptide and nuclear localization signal (NLS) sequences (Figure 1), which was co-expressed with EGFP in BmE-SWU1 to assess whether it could be secreted into the host nucleus. As expected, we found that four NbRBL28 proteins could be located in the BmE-SWU1 cell nucleus (Figure 7c). Although NbRBL28-EGFP fusion protein was located in the host nucleus, NbRBL28 could be secreted into the host nucleus during N. bombycis infection. The result showed that NbRBL28 was not only located in the cytoplasm of the schizont, but was also detected in the host cell nucleus (Figure 7d), which demonstrated that NbRBL28 was a secreted protein targeted to the host cell nucleus.

NbRBL28 Regulates Gene Expressions Involved in Host Cell Cycle
Since NbRBL28 was detected in the infected host cell nucleus, it was likely to account for the gene expression changes triggered by infection. To verify this hypothesis, we performed a transcriptomic analysis of the BmE-SWU1 cells transfected with Nbrbl28::egfpand egfp-containing plasmids. Filtered data were presented in Table S3. To identify which pathways were differently changed, we performed KOG and KEGG pathway analysis. KOG analysis revealed that a number of cell cycle, cell division processes, as well as replication transcription processes, were enriched (Figure 8a). KAGG pathway analysis revealed an enrichment of 11 pathways with p < 0.05 in BmE-SWU1 expressing Nbrbl28::egfp. Genes involved in transcriptional regulation (E2F1 and SDS3) were up-regulated, while a gene (Rad51) functioning in DNA repair was down-regulated (Figure 8b,c). The E2F1 is a transcription factor involved in transformation of cell cycle from G1 phase to S phase [32,33]. The Rad51 participates in the cell cycle, replication and repair [34,35]. In summary,

NbRBL28 Regulates Gene Expressions Involved in Host Cell Cycle
Since NbRBL28 was detected in the infected host cell nucleus, it was likely to account for the gene expression changes triggered by infection. To verify this hypothesis, we performed a transcriptomic analysis of the BmE-SWU1 cells transfected with Nbrbl28::egfpand egfp-containing plasmids. Filtered data were presented in Table S3. To identify which pathways were differently changed, we performed KOG and KEGG pathway analysis. KOG analysis revealed that a number of cell cycle, cell division processes, as well as replication transcription processes, were enriched (Figure 8a). KAGG pathway analysis revealed an enrichment of 11 pathways with p < 0.05 in BmE-SWU1 expressing Nbrbl28::egfp. Genes involved in transcriptional regulation (E2F1 and SDS3) were up-regulated, while a gene (Rad51) functioning in DNA repair was down-regulated (Figure 8b,c). The E2F1 is a transcription factor involved in transformation of cell cycle from G1 phase to S phase [32,33].
The Rad51 participates in the cell cycle, replication and repair [34,35]. In summary, these data indicated that NbRBL28 was positively regulating the expression of host cell genes involved in controlling the cell cycle progression.

Discussion
Ricin B-lectin domain proteins have been identified in bacteria, fungi, plants, invertebrates, and higher animals. Examples include Xylanase in Streptomyces, Ricin in the Castor bean, lactose-binding lectin in earthworms, the mannose-receptor in macrophages and RsA in Rhizoctonia solani [36]. RBL has been identified in most genera of microsporidia, including Anncaliia, Encephalitozoon, Nematocida, and Spraguea [14,37]. Because of its broad distribution and presence in the microsporidia, it is speculated that this gene family predates microsporidia evolution. Encephalitozoon, which has a highly reduced genome, still retains this gene family [38]. We have here identified 52 Nbrbls, which is the largest gene family in N. bombycis. Further study of RBL is helpful to understand the evolution of microsporidia gene and the relationship between microsporidia and its host.
Members of this family form clusters in the genome of microsporidia. In E. cuniculi, four rbls are located on a single syntenic block, and six of the eight rbls of N. ceranae are found in NCER_1015 [14]. In N. bombycis, NBO_0006 was at the core of the gene family, and rbl was most likely the first to appear in this region. In addition, most of the genes appeared in pairs, and there were a large number of duplicate genes. The animal-derived "piggyBac" DNA transposons were found the in genome of N. bombycis. These mobile genetic elements encode functional transposases that are capable of recognizing a specific TTAA motif, which it cleaves to insert itself across different regions of the genome [39]. These results indicated that transposable elements most likely played an important role in mediating the duplication of Nbrbls. It is speculated that N. bombycis has a small number of rbls on a single syntenic block as E. cuniculi in the early stage of evolution, and then gene amplification events such as tandem repeat and fragment repeat in the process of evolution occurred. Gene duplication is very important in the evolution of organisms, and gene duplication and differentiation have been considered as the driving force for a gene to produce new functions. These may suggest that the family would obtain new functions through gene replication and non-synonymous mutation to adapt to the changing living environment.
Our work also showed that evolutionary divergence has also occurred among microsporidia RBL protein genes. First of all, the number of rbl in different microsporidia varies tremendously. There are only 4 rbls in E. cuniculi and 52 rbls in N. bombycis. However, rbl has not been identified in N. parisii genome [11]. Secondly, some members of RBL protein family of microsporidia lost their RBLD. The 52 NbRBLs can be classified into 4 subfamilies with phylogenetic analysis. Subfamily 1 is relatively more conserved as all members have a RBLD and most proteins have SP, indicating that the NbRBLs of subfamily 1 retain an original galactose-binding function. There is big difference among subfamily 1 and other subfamilies, in which some members lost the RBLD. It showed that the sequences of NbRBL varied greatly, so that likely became a multi-functional family. Compared with Ricin B lectin, the motif of NbRBL turned into QxF from QxW, which was also found in the RBLs of Anncaliia algerae [16]. Phenylalanine was replaced by tryptophan, both of which were hydrophobic amino acids. Compared with tryptophan, the molecular weight of phenylalanine is smaller, and the structure becomes simpler. It is suggested that this was a kind of reduction that happened at the amino acid level in microsporidia. Besides, this motif substitution may alter the selectivity of RBLs for specific glycoproteins on host cytoplasm membrane, which are important for the parasite infection.
Previous studies have shown that Nbrbl (identified as Nbrbl03 in our study) was highly transcribed after 42 hpi [15], Our results showed that Nbrbl03 was highly expressed after 12 hpi in BmE-SWU1 cells. We also found that Nbrbl14, Nbrbl46, and Nbrbl51 were also highly expressed at 6 hpi, but some Nbrbls (such as Nbrbl04, Nbrbl05, Nbrbl17, and Nbrbl18, etc.) were expressed at low levels in the infected BmE-SWU1 cells. Interestingly, Nbrbl17 and Nbrbl18 were high level expressed in the infected embryos, which suggested that different NbRBLs may have different biological functions.
Interestingly, NbRBL28 was the first RBL member that was found to be secreted into the host nucleus and likely to modulate the host cell cycle. This modulation model was also reported in other intracellular pathogens [40,41], for instance, T. gondii secrete GRA16, GRA24, ROP16, and TgIST into the host nucleus to interfere with gene expressions [42][43][44][45][46][47][48]. Besides, there are 13 NbRBLs without a SP, of which 9 were predicted to be located in the nucleus ( Table 2), suggesting that these members may regulate the gene expressions of the parasites themselves. Furthermore, it has been reported that lectins have diverse roles in parasites, and can mediate adhesion of the parasite to the host cell [49]. For example, the NbRBL03 was reported to enhance spore adhesion to the host cells [15], and that NbRBL51 is an only member, containing a transmembrane domain, indicating that it is a membrane protein and most likely promotes adhesion too. Similar to the RTB, secreted and transmembrane NbRBLs likely bind to glycoproteins on the host cytoplasm membrane to mediate the adhesion. Therefore, NbRBLs play important and multiple roles during infection and pathogen development.
In summary, we primarily discussed identification, phylogenetic classification, molecular evolution, and gene expression analyses of the NbRBL gene family. The increase of NbRBL genes suggested that certain members have evolved to carry out a larger number of functions to adapt to intracellular life. Therefore, RBL, which is an ideal target, holds significance to the study of microsporidium gene evolution and the analysis of the mechanism of interaction between microsporidium and host.
Author Contributions: T.L. and Z.Z. contributed to conception and design of the study; J.X. and J.L. contributed to experimental analysis; J.X., J.C., and T.L. contributed to data analysis; J.X., T.L., and C.R.V. wrote the first draft of the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by grants from the National Natural Science Foundation of China (31772678 and 31472151) and the Natural Science Foundation of Chongqing, China (cstc2019yszx-jcyjX0010 and cstc2021jcyj-msxmX1003).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The raw data was deposited in GenBank under the BioProject ID PRJNA808047 and BioSample accession SAMN26022686.