The cloning and sequence analysis of grass carp histone H2A variants. (A) The cloning of gcH2A-11 from the gills of grass carp individual 1 infected with F. columnare. (B) The cloning of gcH2A-13 and gcH2A-14~16 from the gills of grass carp individual 2 and 3 infected with F. columnare, respectively. (C) Sequence alignments of gcH2A-4, gcH2A-11 and gcH2A-13~16. The conventional histone H2A region is underlined. (D) Phylogenetic analysis of grass carp histone H2A variants, and histone H2A from other species including shrimp H2A (GenBank accession number: XM_037921631), human H2A.1 (GenBank accession number: M60752), mouse H2A.1 (GenBank accession number: M33988), rat H2A.2 (GenBank accession number: JX661509), human H2A.2 (GenBank accession number: L19779), tongue sole H2A (GenBank accession number: KU904500) and Arabidopsis H2A (GenBank accession number: AK228272).

The effects of grass carp histone H2A variants in bacterial infection. (A–E) No significant effects of gcH2A-1, gcH2A-5, gcH2A-13, gcH2A-14 and gcH2A-15 on the proliferation of F. columnare in vitro. (F–J) The antibacterial effects of gcH2A-2, gcH2A-3, gcH2A-4, gcH2A-11 and gcH2A-16 both at 3 hpi and 6 hpi. (K, L) The antibacterial effects of gcH2A-6 and gcH2A-7 at 6 hpi. Data represented means ± SEM (n=3), and were tested for statistical significance. **p < 0.01; ns, not significant. The asterisk above the bracket indicates statistical significance between the two groups connected by the bracket.

The recombinant expressions of gcH2A-4 and gcH2A-11 in the S. cerevisiae strain. (A) Validation of the recombinant gcH2A-4−PYD1 and gcH2A-11−PYD1 plasmids using BamH I/EcoR I double restriction enzyme digestion. (B) Western Blotting of the cell lysates transformed with the PYD1 empty plasmid. (C) Western Blotting of the cell lysates transformed with PYD1, gcH2A-4−PYD1 or gcH2A-11−PYD1.

Differentially expressed genes and significantly enriched KEGG pathways in grass carp regulated by the engineered S. cerevisiae expressing gcH2A-4 and gcH2A-11. (A) The schematic diagram for the immunized grass carp used for illumina deep sequencing. (B) The number of DEGs regulated by the engineered S. cerevisiae expressing gcH2A-4 and gcH2A-11. (C) The significantly enriched KEGG pathways for up-regulated DEGs in intestines regulated by the engineered S. cerevisiae expressing gcH2A-4 and gcH2A-11. (D) The significantly enriched KEGG pathways for down-regulated DEGs in intestines regulated by the engineered S. cerevisiae expressing gcH2A-4 and gcH2A-11.

The effects of the engineered S. cerevisiae expressing gcH2A-4 on the expression patterns of DEGs involved in PRRs-mediated signaling pathways. (A) The gene cluster for DEGs involved in the NOD-like receptor signaling pathway for the samples from the intestines of grass carp after 7 days of the first immunization. (B) The gene cluster for DEGs involved in the Toll-like receptor signaling pathway for the samples from the intestines of grass carp after 7 days of the first immunization. A color key denotes the gradient scale of gene expression from low (blue) to high (red) degrees. The receptors of NLR and TLR are underlined.

The effects of the engineered S. cerevisiae expressing gcH2A-11 on the expression patterns of DEGs involved in PRRs-mediated signaling pathways. (A) The gene cluster for DEGs involved in the C-type lectin receptor signaling pathway for the samples from the intestines of grass carp after 7 days of the first immunization. (B) The gene cluster for DEGs involved in the Cytosolic DNA-sensing pathway for the samples from the intestines of grass carp after 7 days of the first immunization. (C) The gene cluster for DEGs involved in the RIG-I-like receptor signaling pathway for the samples from the intestines of grass carp after 7 days of the first immunization. (D) The gene cluster for DEGs involved in the Toll-like receptor signaling pathway for the samples from the intestines of grass carp after 7 days of the first immunization. A color key denotes the gradient scale of gene expression from low (blue) to high (red) degrees. The receptors of CLR and TLR are underlined.

Validation of transcriptome data by qRT-PCR. (A) The gene cluster for DEGs involved in the NOD-like receptor signaling pathway for the samples from the intestines of grass carp after 7 days of the first immunization using the engineered S. cerevisiae expressing gcH2A-11. (B) Validation of transcriptome data by qRT-PCR for 11 DEGs involved in the Toll like receptor pathway. Data represented means ± SEM (n=3), and were tested for statistical significance. *p < 0.05; **p < 0.01; ns, not significant.

The effects of the engineered S. cerevisiae expressing gcH2A-4 and gcH2A-11 on the disease resistance of grass carp against F. columnare infection. (A) The schematic diagram for the immunized grass carp used for survival analysis. (B) The effects of the engineered S. cerevisiae expressing gcH2A-4 and gcH2A-11 on the bacteria proliferation of F. columnare Data represented means ± SEM (n=3), and were tested for statistical significance. *p < 0.05; **p < 0.01. (C) The effects of the engineered S. cerevisiae expressing gcH2A-4 and gcH2A-11 on the survival of grass carp in response to F. columnare infection.