Figure 1

Cloning of tyeA gene. M: DL2000 marker. Lane 1-5:The 285bp fragment was amplified from genomic DNA of the wild-type strain HY9901 using primer pairs of tyeAP1-for/tyeAP1-rev (Lane 1, 2, 3, 4, 5:Tm = 56°C, 57°C, 58°C, 59°C, 60°C).
Figure 2

Homology comparison of Vibrio parahaemolyticus HY9901 T3SS Regulator Protein TyeA. Vibrio alginolyticus T3SS regulatory protein TyeA Accession No.ACY41068.1; Vibrio parahaemolyticus T3SS regulatory protein TyeA Accession No.ANB96904.1; Vibrio harveyi T3SS regulatory protein TyeA Accession No.SQA36766.1; Vibrio campbellii T3SS regulatory protein TyeA Accession NO.AQM67418.1; Vibrio tubiashii T3SS regulatory protein TyeA Accession No.WP_004745559.1; Vibrio bivalvicida T3SS regulatory protein TyeA Accession No.OAJ92073.1.
Figure 3

Construction and confirmation of the knockout mutant strain HY9901ΔtyeA. (A) M: DL2000 marker; Lane 1. The 549 bp upstream fragment amplified from genomic DNAs of the strain HY9901 using primer pairs of tyeA-for/tyeA-int-rev; Lane 2. The 543 bp downstream fragment amplified from genomic DNAs of the strain HY9901 using primer pairs of tyeA-int-for / tyeA-rev. (B) M: DL2000 marker; Lane 1. The 1,092 bp fragment amplified from genomic DNAs of HY9901ΔtyeA using primer pairs of tyeA-for/tyeA-rev. (C) M: DL2000 marker; Lane 1–7. The 1,104 bp fragment amplified from genomic DNAs of the HY9901ΔtyeA using primer pairs of tyeA-for/tyeA-rev; Lane 8. The 1,329 bp fragment amplified from genomic DNAs of the wild-type strain HY9901 using primer pairs of tyeA-for/tyeA-rev. (D) M: DL2000 marker; Lane 1–10. The 1,252 bp fragment amplified from genomic DNAs of HY9901ΔtyeA; Lane11. The 1,489 bp fragment amplified from genomic DNAs of the strain HY9901 using primer pairs of tyeA-up / tyeA-down.
Figure 4

Genetic stability detection of HY9901ΔtyeA deletion mutant. M: DL2000 marker, Lane 1. A fragment of 1,489 bp is obtained for HY9901 using primer pairs of tyeA-up/tyeA-down. Lane 2. A fragment of 1,252 bp is obtained for HY9901ΔtyeA using primer pairs of tyeA-up/tyeA-down.
Figure 5

Growth rates of HY9901ΔtyeA and HY9901. Aliquots of cell culture were taken at various time points and measured for cell density at OD600.
Figure 6

Measurement of biofilm by crystal violet ammonium oxalate. *indicates significant difference compared with the control group (p < 0.05). **indicates extremely significant difference compared with the control group (p < 0.01).
Figure 7

Measurement of biofilm by LSCM. (A) HY9901 2.5d diagram. (B) HY9901ΔtyeA 2.5d diagram. (C) HY9901 2d diagram. (D) HY9901ΔtyeA 2d diagram; HY9901 Biofilm thickness:60 ± 10 μm, HY9901ΔtyeA Biofilm thickness:90 ± 20 μm.
Figure 8

Expression of HY9901 and HY9901ΔtyeA T3SS-related genes induced by DMEM. ΔtyeA had significantly increased expression of vscL, vscK, vscO, vopS (p < 0.05), vopN, vscN, and hop (p < 0.01). * indicates significant difference compared with the control group (p < 0.05). ** indicates extremely significant difference compared with the control group (p < 0.01).
Figure 9

Survival in groups vaccinated with HY9901ΔtyeA and PBS following challenge with Vibrio alginolyticus HY9901.
Figure 10

Comparative analysis of the expression of immune-related genes in liver and spleen of zebrafish given the live attenuated vaccine and unvaccinated zebrafish. The head kidney and spleen of grouper were sampled at 1 day before challenge, and the mRNA level of each immune-related gene was normalized to that of β-actin. Bars represent the mean relative expression of three biological replicates and error bars represent standard deviation.
Acknowledgments
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