VdCYC8, Encoding CYC8 Glucose Repression Mediator Protein, Is Required for Microsclerotia Formation and Full Virulence in Verticillium dahliae

Verticillium dahliae is the primary causal agent for Verticillium wilt disease on a diverse array of economically important crops, including cotton. In previous research, we obtained the low-pathogenicity mutant T286 from the T-DNA insertional mutant library of the highly virulent isolate Vd080 derived from cotton. In this study, the target disrupted gene VdCYC8 was identified by TAIL-PCR, encoding a homolog of CYC8 proteins involved in glucose repression. The deletion mutant ΔCYC8 exhibited several developmental deficiencies, including reduced microsclerotia formation, reduced sporulation, and slower growth. Moreover, compared with the wild type strain Vd080, the pathogenicity of strain ΔCYC8 was significantly decreased on cotton seedlings. However, the complementary mutants ΔCYC8-C led to restoration of the wild type phenotype or near wild type levels of virulence on cotton. Interestingly, pathogenicity of the strains was correlated with VdCYC8 gene expression levels in complemented mutants. Gene expression analyses in the wild type strain Vd080, the ΔCYC8-45 strain, and complemented strain ΔCYC8-C26 indicated that VdCYC8 regulates the transcription levels of several genes in V. dahliae that have roles in melanin and production.


Introduction
Verticillium dahliae Kleb. is a soil-borne fungus that causes Verticillium wilt disease on plants worldwide. The melanized microsclerotia produced by V. dahliae can survive over 10 years in the absence of a host, and therefore pose a long-term threat to more than 200 plant species, including several economically important crops such as cotton, potato, strawberry, lettuce [1][2][3][4]. Many isolates of V. dahliae lack host specificity and can be disseminated among different regions and hosts [5]. The microsclerotia germinate under favorable conditions, penetrate

Fungal strains and growth conditions
The virulent defoliating and microsclerotia-forming V. dahliae wild type strain Vd080, derived from typical Verticillium-wilt-symptomatic cotton in Hebei province of China, was used in this study. This isolate and all genetic mutants generated in this study were single-spore purified, and stored in 20% glycerol at -80°C. The fungus was cultured on potato dextrose agar (PDA), and the appropriate antibiotics were added for selection of mutants. The concentration of both hygromycin B and chlorimuron in the media was 50 ug/ml. Conidia production for pathogenicity assays and phenotypic analysis was performed in liquid Czapek-Dox medium as previously described [20].

Gene isolation, cloning and phylogenetic analysis
The virulence-deficient mutant T286 was identified as a single-copy T-DNA integrant [32]. To isolate the target disrupted gene in this current study, a thermal asymmetric interlaced PCR (TAIL-PCR) was conducted with three specific primers on each border [32], and four arbitrary degenerate primers [33]. The specific tertiary PCR products of the appropriate primer combinations were cloned for sequencing. The specific T-DNA insertion site in T286 was identified by comparing the cloned sequences against the available genome sequences of VdLs.17 using BLASTn searches [34].
Thirty-five CYC8 homologous protein sequences from twenty-nine different fungi were identified by BLASTp analysis and downloaded for phylogenetic analysis. Phylogenetic analysis was performed using Mega v.5.1 after multiple alignment of the data by CLUSTAL_X [35], with gaps treated as missing data. Clustering was performed using the neighbor-joining method [36]. Bootstrap analysis was used to evaluate the tree topology of the neighbor-joining data by performing 1000 re-samplings [37].

Vector construction and fungal transformation
A two-step method was used to construct the CYC8 knock out vector, including fusion fragment generation and a gateway cloning reaction. Specific primer pairs CYC8-P1/P3 and CYC8-P2/P4 (Table 1) were designed to amplify the CYC8 upstream (UP) and downstream (DOWN) Vd080 genomic DNA from V. dahliae. Primers Hyg-F/R (Table 1) were designed for amplification of the hygromycin phosphotransferase cassette (HPH, conferring resistance to the antibiotic hygromycin B), obtained from plasmid pCTHyg []. CYC8-P2 and P3 (Table 1) possess adaptors for the reverse complemented sequence of Hyg-F and Hyg-R. Fusion PCR was performed using the DNA fragments of UP, HPH and DOWN together in one reaction. The CYC8 fusion fragment was amplified via nested PCR with primers CYC8-P2/P5 supplementing the adaptor of attB at the 5 0 end (S1A Fig). The binary vector pGKO 2 -Gateway carrying the lethal gene HSVtk was applied for the gateway reaction [38]. With the aid of recombination between attB and attP, the CYC8 fusion fragment was cloned into the binary vector to generate the CYC8 knock out vector, pGKO 2 -CYC8 (S1B Fig).
The binary vector pSULPH-gfp encoding green florescent protein (GFP) and the chlorimuron-resistance marker was used as the backbone for CYC8 complementary vector construction. The CYC8 functional fragment composed of promoter, coding region, and terminator was amplified by the primer pair CYC8COM-F /CYC8COM-R with EcoR1 and AflII recognition sites at the 5' end, respectively ( Table 1). The complementary vector COM-CYC8 was generated by ligation between the backbone vector and functional CYC8 fragment.
Agrobacterium tumefaciens isolate AGL1 carrying the CYC8 knock out binary vector pGKO 2 -CYC8 was transformed into wild-type isolate Vd080, while the complementary vector COM-CYC8, containing the wild type CYC8, was introduced into strain T286 by an Agrobacterium tumefaciens-mediated transformation (ATMT) method described previously [20].

Positive mutants screening and verification
To confirm deletion mutants, two pairs of primers were applied for mutant screening. Hyg-F/R was used to identify successful replacement of target gene VdCYC8 with the hygromycin-resistance cassette. The other pair of tested primers originated from the VdCYC8 coding region; CYC8test-F (5'-GCGTTCGAAAAGGCCAACGA-3') and CYC8test-R (5'-CGTTGGTCATCG AATCGGCG-3') yield a PCR amplicon of 2.1 kb if wild type CYC8 is present. The positive VdCYC8 deletion mutants possessed an HPH-specific band when extracted DNA was amplified using the primer pair Hyg-F/R, but not the wild type VdCYC8 coding region, which could be amplified with primer pair CYC8test-F/R.
For mutant complementation, primers CYC8COM-F/R were used to verify the presence of wild type CYC8, and the chlorimuron-resistance cassette with primers Sul-F (5'-TCGACGTG AGAG CATGCAATTC-3') and Sul-R (5'-GTCGAGGTGCCAACGCC ACAGT-3'). Both genes should be detectable in the genomic DNA of positive complementary mutants. Levels of CYC8 transcriptional expression from Vd080 and complemented mutant strains were assessed by reverse transcription quantitative PCR (RT-qPCR) analysis as described below. The complementation vector carried a GFP marker, the mycelia of the T286 and GFP-transformed Vd080 strains were observed under fluorescence microscopy. Southern blot analysis was conducted to further identify the positive mutants by the presence or absence of the VdCYC8. The genomic DNA of the mutants and Vd080 was digested by EcoR I and separated on a 0.7% agarose gel, and transferred to a nylon membrane (Roche). The probe was amplified from VdCYC8 with the primer CYC8SB-F 5'-GCAGCCTCG GTA CGCAAATC-3' and CYC8SB-R 5'-AGGTACCAGCTCTGTGCG TC-3'. The probe was labeled with digoxigenin (DIG) (Roche, Germany) according to the manufacturer's instructions. Hybridization and detection was performed according to the manufacturer instructions (Roche).

Phenotypic analysis
Developmental and morphological characteristics of both CYC8 deletion (ΔCYC8) and complementary mutants (ΔCYC8-C) were investigated and compared between V. dahliae strain Vd080, transformants of Vd080, and strain T286. The phenotypic analyses included microsclerotia formation, conidia-production, radial growth on PDA, and spore germination rate. The photos of the mutants and wild type were taken post 9 days inoculation using an Olympus microscope, CX 21 (Japan). Phenotypic analyses were performed following methods of Zhang et al. [20] and Tzima et al. [19].

Pathogenicity assays
Verticillium wilt susceptible cotton variety Jimian 11 was used for pathogenicity assays of the genetically transformed mutant strains derived from Vd080. Fungal inoculation, disease investigation, and data analyses were conducted as described previously [39]. A disease index (DI), as previously described [39], was used to evaluate the disease severity of cotton seedlings and pathogenicity of each strain.

Reverse-transcription quantitative Real-Time PCR
To monitor the transcriptional expression profile of melanin and microsclerotia production -related genes in the wild type Vd080 and mutant strains, the gene loci of VDAG_00189, VDAG_00190, VDAG_00184, VDAG_03665, VDAG_03393, VDAG_04954 were selected for quantitative real-time PCR (RT-qPCR) analysis (Table 2) [40]. The expression patterns of VdCYC8 at different stages of development were also assessed by RT-qPCR with V. dahliaeβtubulin as the reference gene. Mycelia of Vd080 were harvested at 4, 8, 12, 16, and 20 days for total RNA isolation.
RNA isolation and cDNA synthesis were performed as mentioned above. RT-qPCR was performed in a LightCycler 480 (Roche, Germany) using SYBR Green I (Bio-Rad, USA). Total volume of PCR reaction was 20 ul, including 10 ul of 2×SYBR Green I SuperMix, 0.2 ul of each primer (Table 2), and 2-ul cDNA of V. dahliae. The reaction profile was performed as an initial 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, appropriate annealing temperature as shown in Table 2 for 15 s and 72°C for 30 s. RNA from each strain was collected from three biological replicates, and three technical replicate reactions were subsequently run for each biological replicate.
RT-qPCR efficiency was determined by 10-fold gradient dilutions of cDNA of each target sequence for standard curve production. Under the optimal annealing temperature, the calculated efficiency of all primers was 90%-105%. β-tublin of V. dahliae was used as reference gene for calibration in all the experiments. Melt curve analyses were carried out to evaluate the primer dimers. Relative expression of each gene was determined from cycle threshold (C T ) values according to the 2 -ΔΔCT method [41].

Cloning, expression pattern and phylogenetic analysis of VdCYC8
In the ATMT insertional mutant strain T286, both T-DNA flanks were successfully identified as VdCYC8 using the appropriate combination of gene-specific primers and four arbitrary degenerate primers ( Fig 1A). The disrupted gene showed high similarity with gene locus VDAG_07052 originating from strain VdLs.17 [34]. The T-DNA was integrated with the first exon of gene VDAG_07052 in T286, as evident by comparison of the sequence to that of the reference genome of strain VdLs.17. VDAG_07052 consists of seven exons and six introns with a full length of 3201 bp, encoding a CYC8 glucose repression mediator protein ( Fig 1B).
The analysis of VdCYC8 relative expression levels at different cultivated periods revealed increased in the early stage and reached maximum at twelve days of growth. However, VdCYC8 expression was subsequently reduced, at remaining time points examined (Fig 2).
Furthermore, phylogenetic analysis indicated that VdCYC8 showed the closest relationship with that of the homolog from a closely related species, Verticillium longisporum and V. alfalfae, followed by Colletotrichum sp.

Selection and verification of VdCYC8 replacement and mutant complementation
VdCYC8-specific upstream (1.1 kb) and downstream (1.2 kb) DNA fragments were amplified from DNA of V. dahliae strain Vd080 with primers CYC8-P1/P3 and CYC8-P2/P4, respectively. The selectable marker gene, encoding hygromycin phosphotransferase (1.8 kb), was amplified from plasmid pCTHyg with primers HygRH-F/R. The VdCYC8 fusion fragment was produced from the fusion of the upstream VdCYC8 flanking sequence, the Hyg resistance marker, and the downstream VdCYC8 flanking sequence fragments. Then, nested PCR was conducted by primers CYC8-P2/P5 with the adaptor of attB at the 5' for the gateway reaction (S1 and S3 Figs). The VdCYC8 knockout vector pGKO 2 -CYC8 carrying the lethal gene HSVtk was generated by gateway reaction (S1B Fig).
Seventeen VdCYC8 deletion mutant strains were selected, single-spored, and cultivated for three generations. Among these, the deletion mutants ΔCYC8-45, ΔCYC8-55 and ΔCYC8-56 were used for verification of VdCYC8 deletion and further investigation (Fig 3D). No ectopic mutants were identified with the lethal gene HSVtk. The Hyg selectable marker cassette was positively identified from DNA extracted from all three knockout mutant strains ΔCYC8-45, ΔCYC8-55 and ΔCYC8-56, but not from Vd080 (Fig 3A and 3B). In contrast to wild type strain Vd080, reverse transcription quantitative PCR analysis revealed lack of gene CYC8 expression in strains ΔCYC8-45, ΔCYC8-55 and ΔCYC8-56 (Fig 3C). This confirmed VdCYC8 replacement with the Hyg selectable marker cassette in the three knockout mutants examined.
The functional complementation fragment (about 4.3kb in all) was amplified from DNA of wild type strain Vd080 using primers CYC8COM-F/CYC8COM-R and integrated into the binary vector pSULPH-gfp. A. tumefaciens isolate AGL1 carrying the CYC8 complementation vector was introduced into the VdCYC8 disruption mutant strain T286. Fifteen positive complemented mutants were generated, which included both the complementation fragment and chlorimuron cassette (Fig 4A and 4B). Afterwards, three genetic-stability and single-sporepurified mutant strains ΔCYC8-C26, ΔCYC8-C30 and ΔCYC8-C36 were selected for target gene expression analysis and fluorescence observation. VdCYC8 was transcribed in all of the tested complemented mutant strains. However, relative expression of VdCYC8 was higher in ΔCYC8-C26 than the other two VdCYC8-complemented mutants, and showed no significant difference with the expression level observed for the wild type strain Vd080 (Fig 4C). Moreover, the mycelia of all the ΔCYC8-C mutants developed normally and exhibited strong green fluorescence under fluorescence microscopy (Fig 5).   VdCYC8 plays critical roles in melanin accumulation, microsclerotia production, and sporulation On PDA, deletion mutants of VdCYC8 exhibited white colonies that were delayed in radial growth, lacked melanin, and produced no microsclerotia (Fig 3D). The average growth rate of VdCYC8 deleted mutants was 2.56±0.17 mm/d, significantly slower than the wild type Vd080 strain at 4.00±0.10 mm/d (Table 3). For the VdCYC8-complemented mutants, fewer microsclerotia appeared than were observed for the wild type strain Vd080, suggesting only partial recovery of the ability of the complemented strain to produce microsclerotia (Fig 5). However, there were no obvious differences in growth rate between the CYC8 complementary mutants and the wild type strain Vd080 (Table 3).
Similar to the T-DNA insertional transformant T286, VdCYC8 deletion strains ΔCYC8-45, ΔCYC8-55, and ΔCYC8-56 exhibited sharply reduced sporulation. Following six days of  growth, the concentration of wild type Vd080 was 6.0 × 10 7 conidia/mL, whereas the densities of VdCYC8 deletion mutant strains ΔCYC8-45, ΔCYC8-55, and ΔCYC8-56 were 5.9 × 10 6 conidia/mL, 5.1 × 10 6 CFU /mL and 1.5 × 10 6 conidia/mL, respectively. Compared with Vd080, the sporulation of the CYC8 deletion mutants was significantly reduced by one order of magnitude. However, in each of the three VdCYC8complemented mutants, ΔCYC8-C26, ΔCYC8-C30, and ΔCYC8-C36, sporulation levels were recovered and the average biomass was in the same order of magnitude as wild type strain Vd080. At 8 14 days of growth, the strains sporulation was consistent among strains, with only slight variation (Table 3). No significant differences in conidia germination were observed between VdCYC8 deletion mutant strains and Vd080 (data not shown).

VdCYC8 is required for virulence on cotton
Pathogenicity assays were conducted to assess the potential contribution of VdCYC8 to the virulence of V. dahliae, strain Vd080. Pathogenicity assays were conducted on cotton seedlings following a root-dip inoculation method [39]. The wild type Vd080, ΔCYC8 and ΔCYC8 complemented strains led to initial symptoms at various times post-inoculation, and with diverse disease progression. Cotton plants infected with the wild type strain Vd080 exhibited typical leaf necrosis and wilting at 7 dpi, and the disease on these plants progressed rapidly. However, the plants inoculated with deletion mutants ΔCYC8-45, ΔCYC8-55 and ΔCYC8-56 did not show noticeable symptoms until 12 dpi. Likewise, the T-DNA insertional mutant strain, T286, caused first visible wilt disease symptoms at 13 dpi. The VdCYC8-complemented strains ΔCYC8-C26, ΔCYC8-C30 and ΔCYC8-C36 caused wilting symptoms at 9 dpi, comparable to the findings observed following inoculation with the wild type strain Vd080. At 24 dpi, 92.2% of the cotton plants inoculated with the wild type Vd080 showed severe symptoms, and a 39.9% mortality rate and a DI value of 48.79±3.11 was recorded. In contrast, for the VdCYC8 deletion mutant strains ΔCYC8-45, ΔCYC8-55, and ΔCYC8-56 reduced pathogenicity with average DI values of 17.7, 23.4 and 18.2 at 24 dpi, respectively. These values represent reductions of 63.7%, 51.8% and 62.7% in strains ΔCYC8-45, ΔCYC8-55, and ΔCYC8-56, respectively, in contrast to the value recorded for the wild type V. dahliae strain Vd080 (DI = 48.8). In addition, there was no significant difference in virulence between deletion mutants ΔCYC8-45, ΔCYC8-55, and ΔCYC8-56 and the original CYC8 insertional mutant strain T286 (Fig 6A and 6B). The reintroduction of VdCYC8 to ΔCYC8 strains restored near wild-type levels of virulence in each of the complemented mutant strains ΔCYC8-C26, ΔCYC8-C30 and ΔCYC8-C36. At 18 dpi, plants infected with VdCYC8-complemented mutant strains showed statistically similar disease levels to those observed when the plants were inoculated with the wild type strain Vd080. At 24 dpi, the VdCYC8-complemented strain ΔCYC8-C26 exhibited strong virulence. Although the strains ΔCYC8-C30 and ΔCYC8-C36 were less virulent than Vd080, the strains ΔCYC8-C30 and ΔCYC8-C36 showed significantly higher DI values than the VdCYC8 deletion mutant strains (Fig 6C and 6D). Interestingly, virulence observed following inoculation of complemented mutant strains was correlated with the VdCYC8 gene expression level (Fig 4C). That is, VdCYC8 gene expression was highest in strain ΔCYC8-C26, relative to VdCYC8, and strain ΔCYC8-C26 showed strongest virulence to cotton seedlings (Fig 6C).
VdCYC8 regulates transcription of several genes involved in melanin production and microsclerotia formation in V. dahliae As mentioned earlier, the ΔVdCYC8 strains of V. dahliae strain Vd080 produced no melanin or microsclerotia, but this phenotype could be partially restored in each of the VdCYC8-complemented mutants. To further investigate the role of VdCYC8 in melanin biosynthesis or microsclerotia biogenesis, the transcriptional expression of VDAG_00189 (encoding laccase), VDAG_00190 (conidial yellow pigment biosynthesis polyketide synthase), VDAG_00184 (amino acid adenylation/polyketide synthase), VDAG_03665 (tetrahydroxy-naphthalene reductase), VDAG_03393 (scytalone dehydratase), and VDAG_04954 (pigment biosynthesis protein) was evaluated in the wild type strain Vd080, the complemented CYC8 mutant strain ΔCYC8-C26, and the knockout mutant ΔCYC8-45. Compared with the expression levels observed in the wild type Vd080 and ΔCYC8-C26 strains, the reduced expression of VDAG_00189, VDAG_00190, VDAG_03665, and VDAG_03393 were each reduced in the absence of ΔVdCYC8, and showed drastic decline in ΔCYC8-45 (Fig 7). However, deletion mutation of CYC8 had no effect on the expression of VDAG_00184 and VDAG_04954 as there were no observed transcriptional alterations for VDAG_00184 and VDAG_04954 among the tested strains (Fig 7).

Discussion
In this study, we examined the function of VdCYC8 of Verticillium dahliae, encoding a homolog of the fungal glucose repression mediator protein CYC8. The homologs of VdCYC8 can be transcriptional repressors, regulating many developmental and metabolic processes in yeast [26,27]. The results of this study indicate that VdCYC8 is involved in microsclerotia formation, conidia production, speed of growth in culture, and virulence on cotton. V. dahliae deletion mutants of CYC8 (ΔCYC8) lost the ability to produce melanin and microsclerotia, and also exhibited decreased sporulation, stunted growth, and sharply reduced pathogenicity on cotton seedlings. Compared with the wild type strain Vd080, the ΔCYC8 strains showed a Analyses of VdCYC8 in V. dahliae 5-day delay in the development of typical Verticillium wilt symptoms. These phenotypic differences in the ΔCYC8 strain were partially or completely restored in complementary mutant strains ΔCYC8-C.
Mirosclerotia are long-term survival structures, and are the primary inoculum for originating Verticillium wilt disease. They are considered to be an important model for exploring the initial penetration and pathogenicity mechanisms in V. dahliae [2,3,42,43]. Entire genomewide expression profiles were conducted to screen differentially expressed genes by comparing cDNA libraries between germinated and non-germinated microsclerotia. One hundred and four genes were expressed exclusively in the germinated microsclerotia, including those encoding the G-protein coupled receptor, cyclopentanone, glucosidase, alcohol dehydrogenase, and others [44]. Conidia serve to transport and disperse the pathogen in the host xylem vessels Transcriptional expression analysis of genes expressed during microsclerotia formation in Verticillium dahliae in the wild-type isolate Vd080, the deletion mutant ΔCYC8-45, and wild type-complemented mutant strain ΔCYC8-C26. The genes highly expressed during microsclerotia formation were previously identified [40], and the primer sets for their amplification in this study are listed in Table 2. The V. dahliae β-tubulin (Bt), amplified using primers VerBt-F/R (see Materials and Methods), was used in relative expression analyses. doi:10.1371/journal.pone.0144020.g007 Analyses of VdCYC8 in V. dahliae [45], procuring nutrients from the host for successful colonization and fungal fitness [46]. Enhancement of desiccation tolerance could prolong survival and accelerate their proliferation within the host [47]. In summary, microsclerotia and conidia are critical propagules for infection and colonization of host plants and are key components of V. dahliae pathogenesis.
In the current study, the VdCYC8 complemented mutant strains exhibited partial restoration of the wild type phenotype. We speculated that the transcriptional expression level was the primary factor affecting the complementation efficiency, and RT-qPCR was conducted to evaluate the VdCYC8 expression level in each complementary mutant. Several protocols are available for gene complementation in V. dahliae including overexpression of target genes [19,48,49], functional copy reintroduction [13,16,17] and exogenous protein expression [47]. Complementary location is random in the deleted mutants without homologous recombination. Most mutants were ectopic insertion instead of specific locus complementation. RT-qPCR was practical in this study to assess the expression level of target genes, and provided data for expression vs phenotype correlation analyses.
The findings herein confirm important roles of VdCYC8 in virulence and development, similar to roles ascribed to the VdCYC8 homolog in morphogenesis and virulence in C. albicans [39]. The defect in VdCYC8 clearly compromised wild type levels of microsclerotial formation, and we speculate that defect directly or indirectly affects virulence in V. dahliae. As reported in other studies, there is an important association between microsclerotia formation and developmental processes that are required for virulence in V. dahliae [19][20][21].
Recent analyses microsclerotia biogenesis and melanin synthesis in V. dahliae by RNA-seq or microarray analysis has revealed numerous target genes that are differentially expressed in microsclerotial and amicrosclerotial cultures [40]. Six candidate genes (VDAG_00189, VDAG_00190, VDAG_00184, VDAG_03665, VDAG_03393, VDAG_04954) involved in melanin synthesis or microsclerotia formation were selected as indicators to investigate the pathways related to VdCYC8. With the absence of VdCYC8, the transcriptional expression of VDAG_00189 (encoding a laccase), VDAG_00190 (conidial yellow pigment biosynthesis polyketide synthase), VDAG_03665 (tetrahydroxy-naphthalene reductase) and VDAG_03393 (scytalone dehydratase) showed significant reductions (Fig 7). This indicated that VdCYC8 was tightly linked with the pathways mentioned above. Interestingly, unlike VDAG_00190, VDAG_00184 (amino acid adenylation/polyketide synthase), which also shared homology with genes from Penicillium marneffei that were involved in melanin biosynthesis [50], was not regulated by VdCYC8 in V. dahliae. Another gene, VDAG_04954, previously identified as up-regulated by RNA sequencing and microarray analysis during microsclerotia development [15,40], was independent of VdCYC8 regulation in this study.
The results presented herein demonstrate the complexity of melanin production and microsclerotia formation in fungi [51,52]. VdCYC8 was involved in several pathways during microsclerotia formation. Further studies are underway to determine the signal transduction pathway of this pivotal, multi-faceted gene in V. dahliae. The roles of VdCYC8 in host penetration, colonization of vascular tissues, and infection will be addressed in future studies, and this knowledge may lead to additional insights on the disruption of signaling for the control of this pathogen.