T6SS Accessory Proteins, Including DUF2169 Domain-Containing Protein and Pentapeptide Repeats Protein, Contribute to Bacterial Virulence in T6SS Group_5 of Burkholderia glumae BGR1

Burkholderia glumae are bacteria pathogenic to rice plants that cause a disease called bacterial panicle blight (BPB) in rice panicles. BPB, induced by B. glumae, causes enormous economic losses to the rice agricultural industry. B. glumae also causes bacterial disease in other crops because it has various virulence factors, such as toxins, proteases, lipases, extracellular polysaccharides, bacterial motility, and bacterial secretion systems. In particular, B. glumae BGR1 harbors type VI secretion system (T6SS) with functionally distinct roles: the prokaryotic targeting system and the eukaryotic targeting system. The functional activity of T6SS requires 13 core components and T6SS accessory proteins, such as adapters containing DUF2169, DUF4123, and DUF1795 domains. There are two genes, bglu_1g23320 and bglu_2g07420, encoding the DUF2169 domain-containing protein in the genome of B. glumae BGR1. bglu_2g07420 belongs to the gene cluster of T6SS group_5 in B. glumae BGR1, whereas bglu_1g23320 does not belong to any T6SS gene cluster in B. glumae BGR1. T6SS group_5 of B. glumae BGR1 is involved in bacterial virulence in rice plants. The DUF2169 domain-containing protein with a single domain can function by itself; however, Δu1g23320 showed no attenuated virulence in rice plants. In contrast, Δu2g07420DUF2169 and Δu2g07420PPR did exhibit attenuated virulence in rice plants. These results suggest that the pentapeptide repeats region of the C-terminal additional domain, as well as the DUF2169 domain, is required for complete functioning of the DUF2169 domain-containing protein encoded by bglu_2g07420. bglu_2g07410, which encodes the pentapeptide repeats protein, composed of only the pentapeptide repeats region, is located downstream of bglu_2g07420. Δu2g07410 also shows attenuated virulence in rice plants. This finding suggests that the pentapeptide repeats protein, encoded by bglu_2g07410, is involved in bacterial virulence. This study is the first report that the DUF2169 domain-containing protein and pentapeptide repeats protein are involved in bacterial virulence to the rice plants as T6SS accessory proteins, encoded in the gene cluster of the T6SS group_5.


Introduction
Burkholderia glumae was first reported in Japan in the 1950s [1]. B. glumae are bacteria pathogenic to rice plants, which cause a disease called bacterial panicle blight (BPB) in rice panicles [2,3]. B. glumae has several virulence factors, such as toxins, proteases, lipases, extracellular polysaccharides, bacterial motility, and bacterial secretion systems [4][5][6][7][8][9][10]. Thus, B. glumae not only cause BPB in rice panicles, but also bacterial wilt in rice stems and various other economically important crops, such as tomatoes, eggplants, hot peppers, and Some T6SS accessory proteins, such as DUF1795, DUF4123, and DUF2169 domaincontaining proteins, are known T6SS adapters that require the loading of a specific effector onto the cognate VgrG for delivery [25]. The DUF2169 domain-containing protein was found to be a T6SS accessory protein in plant pathogenic bacteria, A. tumefaciens; therefore, we focused on only the genes encoding the DUF2169 domain of the T6SS accessory protein [22]. The only two genes encoding the DUF2169 domain in the genome of B. glumae BGR1, bglu_1g23320 and bglu_2g07420, were identified using the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/protein; accessed on 17 August 2021). In particular, bglu_2g07420 was present in the gene cluster of T6SS group_5 in B. glumae BGR1 ( Figure 1A). However, bglu_1g23320 was not included in the gene cluster of any T6SS group and existed independently as a separate entity ( Figure 1B). The bglu_1g23320 gene is a hypothetical protein (WP_015876299.1) composed of 362 amino acids, whereas bglu_2g07420 is a hypothetical protein (WP_012733555.1) composed of 878 amino acids. Functional analysis of the proteins encoded by the two genes was performed using the InterPro database (http://www.ebi.ac.uk/interpro; accessed on 17 August 2021) ( Table 1). The bglu_1g23320 gene encodes only the DUF2169 domain, whereas the bglu_2g07420 gene encodes the DUF2169 domain at the N-terminus and the pentapeptide repeats region at the C-terminus ( Figure 2). Additionally, the non-annotated gene, bglu_2g07410, located adjacent to bglu_2g07420, encoding the DUF2169 domain-containing protein, was also analyzed with the InterPro database. The bglu_2g07410 gene, which is located downstream of the bglu_2g07420 gene, was evaluated as a pentapeptide repeats protein (WP_012733554.1), encoding only the pentapeptide repeats region consisting of 356 amino acids ( Figure 1A and Table 1). We assumed that the bglu_2g07420 gene, which is present in the gene cluster of T6SS group_5, and the other gene of bglu_1g23320, which is not present in any T6SS group, are the major putative T6SS accessory proteins involved in the function of T6SS group_5 in B. glumae BGR1. Furthermore, we assumed that the bglu_2g07410 gene encoding the pentapeptide repeats region, similarly to the bglu_2g07420 gene with its pentapeptide repeats region, is involved in the function of T6SS group_5 as a putative T6SS accessory protein.
BGR1 was found to be affected by the loss of the pentapeptide repeats region in bglu_2g07420 and bglu_2g07410. bglu_2g07410 encodes the pentapeptide repeats protein with only the pentapeptide repeats region. Our findings show that bglu_2g07420 and bglu_2g07410, belonging to the gene cluster of T6SS group_5 in B. glumae BGR1, encode the DUF2169 domain-containing protein and pentapeptide repeats protein, respectively, and contribute to the bacterial virulence to rice plants as the T6SS-associated proteins.

Investigation and Analysis of the Genes Containing the DUF2169 Domain and Its Adjacent Gene in the Genome of Burkholderia glumae BGR1
Some T6SS accessory proteins, such as DUF1795, DUF4123, and DUF2169 domaincontaining proteins, are known T6SS adapters that require the loading of a specific effector onto the cognate VgrG for delivery [25]. The DUF2169 domain-containing protein was found to be a T6SS accessory protein in plant pathogenic bacteria, A. tumefaciens; therefore, we focused on only the genes encoding the DUF2169 domain of the T6SS accessory protein [22]. The only two genes encoding the DUF2169 domain in the genome of B. glumae BGR1, bglu_1g23320 and bglu_2g07420, were identified using the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/protein; accessed on 17 August 2021). In particular, bglu_2g07420 was present in the gene cluster of T6SS group_5 in B. glumae BGR1 ( Figure 1A). However, bglu_1g23320 was not included in the gene cluster of any T6SS group and existed independently as a separate entity ( Figure 1B). The bglu_1g23320 gene is a hypothetical protein (WP_015876299.1) composed of 362 amino acids, whereas bglu_2g07420 is a hypothetical protein (WP_012733555.1) composed of 878 amino acids. Functional analysis of the proteins encoded by the two genes was performed using the InterPro database (http://www.ebi.ac.uk/interpro; accessed on 17 August 2021) ( Table 1). The bglu_1g23320 gene encodes only the DUF2169 domain, whereas the bglu_2g07420 gene encodes the DUF2169 domain at the N-terminus and the pentapeptide repeats region at the C-terminus ( Figure 2). Additionally, the non-annotated gene, bglu_2g07410, located adjacent to bglu_2g07420, encoding the DUF2169 domain-containing protein, was also analyzed with the InterPro database. The bglu_2g07410 gene, which is located downstream of the bglu_2g07420 gene, was evaluated as a pentapeptide repeats protein (WP_012733554.1), encoding only the pentapeptide repeats region consisting of 356 amino acids ( Figure 1A and Table 1). We assumed that the bglu_2g07420 gene, which is present in the gene cluster of T6SS group_5, and the other gene of bglu_1g23320, which is not present in any T6SS group, are the major putative T6SS accessory proteins involved in the function of T6SS group_5 in B. glumae BGR1. Furthermore, we assumed that the bglu_2g07410 gene encoding the pentapeptide repeats region, similarly to the bglu_2g07420 gene with its pentapeptide repeats region, is involved in the function of T6SS group_5 as a putative T6SS accessory protein. Figure 1. Genetic organization of T6SS group_5, containing bglu_2g07420 encoding the DUF2169containing protein and bglu_2g07410 encoding pentapeptide repeats protein (A), and bglu_1g23320 encoding the DUF2169-containing protein (B) in Burkholderia glumae BGR1. The genes are indicated by the locus ID (e.g., bglu_2g07410) and are to scale. The genes marked in gray encode the DUF2169 domain-containing proteins and pentapeptide repeats protein. To determine whether the DUF2169 domain encoded by bglu_1g23320 and bglu_2g07420 is involved in the functional role of T6SS group_5 in bacterial virulence, causing bacterial panicle blight, rice plants at the flowering stage were infected with wildtype B. glumae BGR1 and the mutants Δu1g23320 and Δu2g07420DUF2169 (Figure 3). At  To investigate the functions of putative T6SS accessory proteins containing the DUF2169 domain, we constructed markerless deletion mutants targeting the DUF2169 domain of bglu_1g23320 and bglu_2g07420 genes via homologous recombination. Single markerless deletion mutants, called ∆u2g07420DUF2169 and ∆u1g23320, were generated via the deletion of only the DUF2169 domain of bglu_2g07420 and bglu_1g23320, respectively (Supplementary Tables S1 and S2). To complement the phenotypes of ∆u2g07420DUF2169 and ∆u1g23320, the respective complementation strains, ∆u2g07420DUF2169-C and ∆u1g23320-C, were generated.
To investigate whether the pentapeptide repeats region encoded in bglu_2g07410 and C-terminus of bglu_2g07420 affect the function of T6SS group-5 as putative T6SS accessory proteins, the pentapeptide repeats region of both genes was deleted to construct markerless deletion mutants. Single markerless deletion mutants, called ∆u2g07420PPR and ∆u2g07410, were generated by deleting the pentapeptide repeats region of bglu_2g07420 and bglu_2g07410, respectively (Supplementary Tables S2 and S3). To complement the phenotypes of ∆u2g07420PPR and ∆u2g07410, the respective complementation strains, ∆u2g07420PPR-C and ∆u2g07420-C, were generated. A double markerless deletion mutant, ∆u2g07410-20, was generated to determine whether two separate pentapeptide repeats regions independently contributed to the function of T6SS group_5. To complement the phenotype of ∆u2g07410-20, the respective complementation strain, ∆u2g07410-20-C, was generated.

The DUF2169 Domain Encoded by the N-terminus of bglu_2g07420 inside the Gene Cluster of T6SS Group_5 Is Involved in Bacterial Virulence in Rice Plants
To determine whether the DUF2169 domain encoded by bglu_1g23320 and bglu_2g07420 is involved in the functional role of T6SS group_5 in bacterial virulence, causing bacterial panicle blight, rice plants at the flowering stage were infected with wild-type B. glumae BGR1 and the mutants ∆u1g23320 and ∆u2g07420DUF2169 (Figure 3). At 8 days post-inoculation (dpi), most of the wild-type BGR1-inoculated panicles had developed blight disease symptoms (severity, 0-5), with a disease severity score of 4.38 ± 0.079 for wild-type BGR1 ( Figure 3B). In contrast, ∆u2g07420DUF2169 exhibited attenuated virulence, with a disease severity score of 2.73 ± 0.134 ( Figure 3B). The restored strain, 2g07420DUF169-C, completely recovered its virulence, with a disease severity score of 4.27 ± 0.26 ( Figure 3B). Rice plants were also infected with the ∆u1g23320 mutant to determine whether the bglu_1g23320 gene contributed to bacterial virulence. However, the disease severity score of ∆u1g23320 was 4.26 ± 0.046 ( Figure 3B). The deletion of the DUF2169 domain in bglu_1g23320 caused almost no difference in pathogenicity from wild-type BGR1, although the deletion of the DUF2169 domain in bglu_2g07420 differed from that of wild-type BGR1 ( Figure 3). Thus, only the DUF2169 domain (WP_012733555.1), which is encoded by bglu_2g07420 in the gene cluster of T6SS group_5, is involved in bacterial virulence to rice plants. Plants 2022, 10, x FOR PEER REVIEW 6 of 15

The Pentapeptide Repeats Region Encoded by bglu_2g07410 and the C-terminus of bglu_2g07420 inside the Gene Cluster of T6SS Group_5 Is Involved in Bacterial Virulence to Rice Plants
To determine whether only the DUF2169 domain encoded by the N-terminus of bglu_2g07420 is involved in the functional role of T6SS group_5, rice plants at the flowering stage were infected with Δu2g07420PPR, in which the C-terminal pentapeptide repeats region is deleted. At 8 dpi, most of the wild-type BGR1-inoculated panicles had developed blight disease symptoms (severity, 0-5), with a disease severity score of 4.38 ± 0.079 for wild-type BGR1 ( Figure 4B). In contrast, Δu2g07420PPR displayed attenuated virulence, with a disease severity score of 3.05 ± 0.022 ( Figure 4B). To determine whether the pentapeptide repeats region is also involved in bacterial virulence, rice plants were infected with Δu2g07410, in which bglu_2g07410 was deleted downstream of bglu_2g07420. Δu2g07410 also showed attenuated virulence, with a disease severity score of 2.59 ± 0.114 ( Figure 4B). Δu2g07410-20 exhibited attenuated virulence, with a disease severity score of 1.85 ± 0.091 ( Figure 4B), as did Δu2g07410-20 ( Figure 4B). The restored strains, namely, Δu2g07420PPR-C, Δu2g07410-C, and Δu2g07410-20-C, completely recovered their virulence, with disease severity scores of 4.32 ± 0.279, 4.26 ± 0.193, and 4.18 ± 0.179, respectively ( Figure 4). The levels of attenuated virulence by single mutants, Δu2g07420 and Δu2g07410, were similar at the reproductive and flowering stages of rice plants (Figures 4 and 5). Thus, the pentapeptide repeats regions of bglu_2g07420 and bglu_2g07410 also contribute to bacterial virulence in rice plants. However, the results of in vivo pathogenicity assays at the reproductive stage showed that the bacterial virulence of Δu2g07410-20, in which both genes were deleted, was more attenuated than that of single mutants (Figure 4). The mean disease severity score of Δu2g07410-20 and the mean disease severity scores of Δu2g07410 or Δu2g07420 were compared using the least significant difference at p < 0.001 according to Tukey's HSD test.  To determine whether only the DUF2169 domain encoded by the N-terminus of bglu_2g07420 is involved in the functional role of T6SS group_5, rice plants at the flowering stage were infected with ∆u2g07420PPR, in which the C-terminal pentapeptide repeats region is deleted. At 8 dpi, most of the wild-type BGR1-inoculated panicles had developed blight disease symptoms (severity, 0-5), with a disease severity score of 4.38 ± 0.079 for wild-type BGR1 ( Figure 4B). In contrast, ∆u2g07420PPR displayed attenuated virulence, with a disease severity score of 3.05 ± 0.022 ( Figure 4B). To determine whether the pentapeptide repeats region is also involved in bacterial virulence, rice plants were infected with ∆u2g07410, in which bglu_2g07410 was deleted downstream of bglu_2g07420. ∆u2g07410 also showed attenuated virulence, with a disease severity score of 2.59 ± 0.114 ( Figure 4B). ∆u2g07410-20 exhibited attenuated virulence, with a disease severity score of 1.85 ± 0.091 ( Figure 4B), as did ∆u2g07410-20 ( Figure 4B). The restored strains, namely, ∆u2g07420PPR-C, ∆u2g07410-C, and ∆u2g07410-20-C, completely recovered their virulence, with disease severity scores of 4.32 ± 0.279, 4.26 ± 0.193, and 4.18 ± 0.179, respectively ( Figure 4). The levels of attenuated virulence by single mutants, ∆u2g07420 and ∆u2g07410, were similar at the reproductive and flowering stages of rice plants (Figures 4 and 5). Thus, the pentapeptide repeats regions of bglu_2g07420 and bglu_2g07410 also contribute to bacterial virulence in rice plants. However, the results of in vivo pathogenicity assays at the reproductive stage showed that the bacterial virulence of ∆u2g07410-20, in which both genes were deleted, was more attenuated than that of single mutants (Figure 4). The mean disease severity score of ∆u2g07410-20 and the mean disease severity scores of ∆u2g07410 or ∆u2g07420 were compared using the least significant difference at p < 0.001 according to Tukey's HSD test.

Discussion
T6SS has evolved into the most complex secretory pathway because of its key role during interactions with the surroundings [29]. T6SS is known to have two distinct functional features: a prokaryotic targeting system with antibacterial effects manifesting as bacterial interactions, and a eukaryotic targeting system demonstrating bacterial virulence as eukaryotic interactions. T6SS is a proteinaceous machine with 13 core components. The core genes of T6SS are organized into genomic clusters. In addition to the core components, the gene cluster of T6SS additionally encodes accessory genes functionally associated with T6SS [20,[30][31][32][33][34]. These accessory proteins are either involved in the secretion of T6SS-dependent effectors or regulate the assembly and activity of T6SS [35]. The functions of most T6SS accessory proteins have not been identified. In particular, the protein comprising the DUF2169 domain exists as a single-domain protein with only the DUF2169 domain; however, some are multi-domain proteins with additional domains as small portions at the C-terminus [36]. The most common additional domain of the DUF2169 domain-containing protein is the pentapeptide repeats region, which has unknown functions [36].
The DUF2169 domain-containing protein of a single-domain protein can be involved in the functional role of T6SS as an adapter, even if there is no additional domain at the Cterminus. For example, a single DUF2169 domain-containing protein encoded by atu3641 in Agrobacterium tumefaciens C58 is a T6SS adapter associated with Tde2, an effector of T6SS, and contributes to the secretion of Tde2 [22].
B. glumae BGR1 has a DUF2169 domain-containing protein encoded by two genes, bglu_1g23320 and bglu_2g07420, on its genome. These genes are located downstream of the vgrG. The genes which encode T6SS accessory proteins, such as adaptors, are often found downstream of vgrG [27]. bglu_1g23320 encodes a single-domain protein with only the DUF2169 domain. The bglu_1g23320 gene was not included in any T6SS gene cluster of B. glumae BGR1 (Figure 1). Some T6SS accessory genes and its upstream gene, vgrG, are located outside the T6SS gene cluster and borrow the core components of T6SS [37]. However, Δu1g23320 did not show attenuated virulence in rice plants (Figure 3). T6SS adapters, such as the DUF2169 domain-containing protein, enable secretion by loading specific

Discussion
T6SS has evolved into the most complex secretory pathway because of its key role during interactions with the surroundings [29]. T6SS is known to have two distinct functional features: a prokaryotic targeting system with antibacterial effects manifesting as bacterial interactions, and a eukaryotic targeting system demonstrating bacterial virulence as eukaryotic interactions. T6SS is a proteinaceous machine with 13 core components. The core genes of T6SS are organized into genomic clusters. In addition to the core components, the gene cluster of T6SS additionally encodes accessory genes functionally associated with T6SS [20,[30][31][32][33][34]. These accessory proteins are either involved in the secretion of T6SSdependent effectors or regulate the assembly and activity of T6SS [35]. The functions of most T6SS accessory proteins have not been identified. In particular, the protein comprising the DUF2169 domain exists as a single-domain protein with only the DUF2169 domain; however, some are multi-domain proteins with additional domains as small portions at the C-terminus [36]. The most common additional domain of the DUF2169 domain-containing protein is the pentapeptide repeats region, which has unknown functions [36].
The DUF2169 domain-containing protein of a single-domain protein can be involved in the functional role of T6SS as an adapter, even if there is no additional domain at the C-terminus. For example, a single DUF2169 domain-containing protein encoded by atu3641 in Agrobacterium tumefaciens C58 is a T6SS adapter associated with Tde2, an effector of T6SS, and contributes to the secretion of Tde2 [22].
B. glumae BGR1 has a DUF2169 domain-containing protein encoded by two genes, bglu_1g23320 and bglu_2g07420, on its genome. These genes are located downstream of the vgrG. The genes which encode T6SS accessory proteins, such as adaptors, are often found downstream of vgrG [27]. bglu_1g23320 encodes a single-domain protein with only the DUF2169 domain. The bglu_1g23320 gene was not included in any T6SS gene cluster of B. glumae BGR1 (Figure 1). Some T6SS accessory genes and its upstream gene, vgrG, are located outside the T6SS gene cluster and borrow the core components of T6SS [37]. However, ∆u1g23320 did not show attenuated virulence in rice plants (Figure 3). T6SS adapters, such as the DUF2169 domain-containing protein, enable secretion by loading specific effectors into cognate VgrG (Valine glycine repeat G) without affecting the function of T6SS [22,27,35]. Therefore, the DUF2169 domain-containing protein encoded by bglu_1g23320, which is not included in any T6SS gene cluster of B. glumae BGR1, is not involved in a specific effector that directly causes pathogenicity in rice plants, or may remain as a non-functional T6SS accessory protein.
In contrast to bglu_1g23320, bglu_2g07420 encodes a multi-domain protein with the DUF2169 domain and pentapeptide repeats region as an additional domain at the Cterminus. Additionally, bglu_2g07420 is included in the T6SS group_5 gene cluster of B. glumae BGR1. bglu_2g07420 encodes a DUF2169 domain-containing protein and is present in the gene cluster of T6SS group_5; therefore, a DUF2169 domain-containing protein encoded by bglu_2g07420 can be considered as a T6SS accessory protein of T6SS group_5 in B. glumae BGR1. The T6SS group_5 of B. glumae BGR1 is involved in bacterial virulence in rice plants [3]. In addition, the T6SS-5 gene cluster of B. pseudomallei and B. thailandensis belonging to the eukaryotic system has tagA/B-5, which encodes the DUF2169 domain-containing protein [38]. Additionally, TagA/B-5 of B. thailandensis is a multidomain protein composed of the DUF2169 domain and pentapeptide repeats region as an additional domain at the C-terminus, which is essential for full virulence in multinucleated giant cells [39]. Thus, the DUF2169 domain-containing protein encoded by bglu_2g07420 was also hypothesized to be essential for bacterial virulence as a T6SS accessory protein of the T6SS group_5 in B. glumae BGR1.
The single-domain protein containing DUF2169 can function by itself; therefore, ∆u2g07420DUF2169 was generated by deleting only DUF2169 of the DUF2169 domaincontaining protein without causing any problem in the frameshift of bglu_2g07420. Furthermore, an in vivo pathogenicity assay was performed with this strain. The attenuated virulence of ∆u2g07420DUF2169 indicated that the DUF2169 domain of the DUF2169 domain-containing protein encoded by bglu_2g07420 is required for bacterial virulence in rice plants. ∆u2g07420PPR was generated to determine whether the DUF2169 domaincontaining protein encoded by bglu_2g07420 can fully function as a T6SS accessory protein even in the absence of an additional domain. An in vivo pathogenicity assay was also performed using this strain. Similarly to ∆u2g07420DUF2169, the attenuated virulence of ∆u2g07420PPR indicated that the DUF2169 domain-containing protein encoded by bglu_2g07420 requires not only the DUF2169 domain, but also the additional C-terminal domain, the pentapeptide repeats region, to enable bacterial virulence in rice plants. Therefore, the DUF2169 domain-containing protein encoded by bglu_2g07420 is a functional T6SS accessory protein consisting of multiple domains having the DUF2169 domain and an additional C-terminal pentapeptide repeats region.
bglu_2g07410, located downstream of bglu_2g07420 which encodes the additional C-terminal domain of the pentapeptide repeats region, encodes only the pentapeptide repeats region. The protein containing only the pentapeptide repeats region might also contribute to bacterial virulence in rice plants with the DUF2169 domain-containing protein having an additional C-terminal pentapeptide repeats region. Indeed, ∆u2g07410 also showed attenuated virulence in rice plants. The attenuated virulence of ∆u2g07410 indicates that the pentapeptide repeats protein, encoded by bglu_2g07410, also contributes to bacterial virulence in rice plants. Pentapeptide repeats proteins are widely distributed in both prokaryotes and eukaryotes [40]. These proteins may be responsible for targeting, or for structural functions, rather than enzymatic activity [40]. These proteins have a superhelical structure and can participate in protein-protein interactions without conferring biological functions [40]. For example, the pentapeptide repeats regions of MfpA and Qnr have a superhelical structure, mimicking the B-form of DNA and inhibiting DNA gyrase, conferring resistance to fluoroquinolone [41][42][43][44]. However, the functions or roles of pentapeptide repeats proteins are not fully understood.
Interestingly, the type III secretion system-dependent effector, PipB2, in Salmonella spp. contains the pentapeptide repeats region, which directly interacts with kinesin-1 and contributes to the formation of Salmonella-induced filaments. However, the exact function and role of the pentapeptide repeats region in PipB2 is also unclear [41][42][43]. Interestingly, in the results of the in vivo pathogenicity assay at the reproductive stage, the severely attenuated virulence by ∆u2g07410-20 was shown to be a synergistic effect of the two T6SS accessory proteins encoded by bglu_2g07410 and bglu_2g07420. B. glumae BGR1 mainly causes BPB as a serious symptom in the reproductive stage of rice plants, but can also cause diseases at any developmental stage, such as seed rot, stunting in seedlings, and sheath rot in the vegetative stage. Neopseudocercosporella capsellae, a plant pathogen, causes disease in canola, and the incidence and the severity of the disease are determined by the growth stage of the host plant in which this pathogen invasion occurs [45]. Therefore, the results of the in vivo pathogenicity assay at the reproductive stage show that B. glumae BGR1 induces BPB, with distinct functions of these two T6SS accessory proteins involved in bacterial virulence at the reproductive stage.
In the current study, we obtained genes encoding the DUF2169 domain from the genome of B. glumae BGR1 and investigated whether the genes encoding the DUF2169 domain-containing proteins consisting of a single domain or multi-domain affect bacterial virulence to rice plants. Of the two genes encoding the DUF2169 domain-containing protein, bglu_1g23320 and bglu_2g07420, only the latter was involved in bacterial virulence. We discovered two genes encoding the T6SS accessory proteins in the gene cluster of T6SS group_5 in B. glumae BGR1: bglu_2g07420 and bglu_2g07410. Our results show that the DUF2169 domain-containing protein encoded in bglu_2g074720 is a multi-domain protein that requires both the DUF2169 domain and the pentapeptide repeats region to contribute to bacterial virulence. T6SS accessory protein, having a DUF2169 domain, is a well-known T6SS adaptor. The DUF2169 domain-containing protein encoded by bglu_2g07420 could be considered as a potential T6SS adaptor; however, additional experimental evidence, such as interactions with VgrG or specific T6SS-dependent effectors, is required to confirm its function as a T6SS adaptor. Furthermore, we showed that another T6SS accessory protein, encoded by bglu_2g07410 located downstream of bglu_2g07420, is involved in bacterial virulence as the pentapeptide repeats protein. To the best of our knowledge, this is the first report on the identification of the T6SS accessory proteins, the DUF2169 domain-containing protein, and the pentapeptide repeats protein, belonging to T6SS group_5 of B. glumae BGR1 and involved in bacterial virulence. Novel T6SS accessory proteins involved in the bacterial virulence of B. glumae BGR1 can be usefully employed to understand the functional activity of T6SS.

Bacterial Strains, Plasmids, and Growth Conditions
All bacterial strains and plasmids used in this study are detailed in Table 2. The wild-type BGR1, mutant strains, and E. coli were cultured on Luria Bertani (LB) agar and in broth medium at 37 • C with shaking at 200 rpm. The growth curve of bacterial strains was determined every 2 h by measuring the optical density at 600 nm (OD 600 ) using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). When required, LB media were supplemented with antibiotics at concentrations of 100 µg/mL (rifampicin) and 50 µg/mL (kanamycin).

Construction of Markerless Deletion Mutants and Their Complemetation Strains in B. glumae BGR1
All primer pairs used for the construction of deletion mutants are shown in Supplementary Table S3. Plasmids, including pK18u1g23320, pK18u2g07410, pK18u2g07420DUF2169, and pk18u2g07420PPR, harboring portions of the target gene upstream (L fragment) and downstream (R fragment), were prepared. The L and R fragments were designed according to the Gibson assembly reaction. The L and R fragments were ligated by secondary PCR using LF and RR primer pairs. To construct pK18u1g23320, the ligated LR fragments and pK18mobsacB were digested with BamHI-HindIII (NEB, Ipswich, MA, USA). To construct pK18u2g07410, the ligated LR fragments and pK18mobsacB were digested with EcoRI and HindIII. To construct pK18u2g07420DUF2169, the ligated LR fragments and pK18mobsacB were digested with EcoRI and HindIII. To construct pK18u2g07420PPR, the ligated LR fragments and pK18mobsacB were digested with BamHI and HindIII. Competent E. coli DH5a cells were transformed with recombinant plasmids and selected in kanamycin-containing media. Recombinant plasmids were transformed into E. coli S17-1 cells. Recombinant plasmids were transformed into B. glumae BGR1 by conjugation through co-culture with E. coli S17-1 on LB agar plates. The first crossover was selected on media containing kanamycin (60 µg/mL) and rifampicin (100 µg/mL). The first crossover of colonies was confirmed by PCR using the primer pairs of genes, _UP_F and pK18_DOWN_R. The selected cells underwent a second crossover in LB broth containing kanamycin (60 µg/mL). The second crossover in the cells was selected on LB agar, containing 30% sucrose and rifampicin (100 µg/mL). Finally, single deletions of bglu_1g23320, bglu_2g07410, the DUF2169 domain region, and the pentapeptide repeats region of bglu_2g07420 gene were confirmed by PCR using the primer sets for gene_UP_F and gene_DOWN_R. The double deletion mutant, ∆2g07410-20, was constructed based on single ∆2g07410 deletion mutants using the above-mentioned methods.
For complementation, the entire open reading frame of bglu_2g07410 and bglu_2g07420 genes was amplified by PCR using the primer pairs Cgene_F and Cgene_R. The amplified DNA fragments were cloned into the broad-host-range expression vector, pBBR1MCS2. The cloned vectors, pBu2g07410 and/or pBu2g07420, were conjugated into single and double deletion mutant strains by co-culturing with S17-1. Finally, complementation strains of single and double deletion mutants were selected on media containing rifampicin (100 µg/mL) and kanamycin (60 µg/mL) and were confirmed by PCR using primer pairs of pB_UP_F and pB_DOWN_R.

In Vivo Pathogenicity Assay at the Vegetative and Reproductive Stages of Rice Plants
To examine the bacterial virulence of putative T6SS accessory proteins in B. glumae BGR1 at the vegetative and reproductive stages of rice plants, cultured bacterial cells in the mid-logarithmic phase were harvested by centrifugation, washed, and resuspended in distilled water. Thereafter, the optical density of the bacterial suspensions was adjusted to OD 600nm = 0.8. The rice plants (Oryza sativa L. Saeilmi) used in this experiment were grown under greenhouse conditions (average 30 • C in the day, and 25 • C at night). Subsequently, the stems of vegetative-stage rice plants were inoculated with bacterial suspensions using a syringe and grown for 8 days. Disease severity was observed in the inoculated area. Suspensions for each bacterial strain were prepared and adjusted to OD 600nm = 0.5 to confirm bacterial panicle blight disease at the rice reproductive stage. At the flowering stage, the rice panicles were inoculated via dipping into 50 mL bacterial suspensions for 1 min. At 8 dpi, the disease severity of the rice panicles in individual rice plants was evaluated using the following scale: 0-healthy panicle; 1-0-20% diseased panicle; 2-20-40% diseased panicle; 3-40-60% diseased panicle; 4-40-80% diseased panicle; 5-80-100% diseased panicle. Disease severity was calculated using the following formula: disease severity = Σ (number of samples per rating × rating value)/total number of panicles.

Statistical Analysis
All experiments were conducted at least three times, with at least three replicates. Analyses of the disease severity scores were conducted with at least three replicates of individual rice plants. The mean disease severity scores between two groups were compared using the least significant difference test according to Tukey's HSD assay.