Comprehensive Analysis of Respiratory Burst Oxidase Homologs (Rboh) Gene Family and Function of GbRboh5/18 on Verticillium Wilt Resistance in Gossypium barbadense

Abstract

Respiratory burst oxidase homologs (Rbohs) play a predominant role in reactive oxygen species (ROS) production, which is crucial in plant growth, differentiation, as well as their responses to biotic and abiotic stresses. To date, however, there is little knowledge about the function of cotton Rboh genes. Here, we identified a total of 87 Rbohs from five sequenced Gossypium species (the diploids Gossypium arboreum, Gossypium raimondii, and Gossypium australe, and the allotetraploids Gossypium hirsutum and Gossypium barbadense) via BLAST searching their genomes. Phylogenetic analysis of the putative 87 cotton Rbohs revealed that they were divided into seven clades. All members within the same clade are generally similar to each other in terms of gene structure and conserved domain arrangement. In G. barbadense, the expression levels of GbRbohs in the CladeD were induced in response to a fungal pathogen and to hormones (i.e., jasmonic acid and abscisic acid), based upon which the main functional member in CladeD was discerned to be GbRboh5/18. Further functional and physiological analyses showed that the knock-down of GbRboh5/18 expression attenuates plant resistance to Verticillium dahliae infection. Combined with the molecular and biochemical analyses, we found less ROS accumulation in GbRboh5/18-VIGS plants than in control plants after V. dahliae infection. Overexpression of GbRboh5/18 in G. barbadense resulted in more ROS accumulation than in control plants. These results suggest that GbRboh5/18 enhances the cotton plants' resistance against V. dahliae by elevating the levels of ROS accumulation. By integrating phylogenetic, molecular, and biochemical approaches, this comprehensive study provides a detailed overview of the number, phylogeny, and evolution of the Rboh gene family from five sequenced Gossypium species, as well as elucidating the function of GbRboh5/18 for plant resistance against V. dahliae. This study sheds fresh light on the molecular evolutionary properties and function of Rboh genes in cotton, and provides a reference for improving cotton's responses to the pathogen V. dahliae.

Keywords: GbRboh5/18; Gossypium; V. dahliae; reactive oxygen species (ROS); respiratory burst oxidase homologs (Rbohs).

FIGURE 1

Phylogenetic analysis of Rbohs in cotton and Arabidopsis. A phylogenetic tree of Rboh proteins from G. arboreum, G. raimondii, G. australe, G. hirsutum, G. barbadense, and A. thaliana. The full-length amino acid sequences of the Rboh proteins were aligned using ClustalX in MEGA7.0. The unrooted tree was generated by the neighbor-joining (NJ) method (n = 1000 bootstraps). Clades are distinguished by different colors. Cotton Rbohs are colored clade specifically and the Arabidopsis Rbohs are in black. Different-colored solid circles indicate genes in different Gossypium species or Arabidopsis.

FIGURE 2

Characterization of the 87 Rbohs in five Gossypium species. The characteristics include the homologous relationship, conserved domain location, and exon-intron structure. (A) Phylogenetic relationships among the Rbohs in five Gossypium species. Rbohs in same clade are indicated by the same color. Different-colored solid circles indicate genes in different Gossypium species. (B) Conserved domain architecture of cotton Rboh proteins. Motifs were identified through a MEME analysis (http://meme-suite.org/) and protein length was estimated via the scale at the bottom. (C) Exon-intron organization analyses of cotton Rboh genes. The gene length was estimated by the scale at the bottom through gene structure analyses (GSDS: http://gsds.cbi.pku.edu.cn/). Green boxes indicate 5′- or 3′-untranslated regions. Exons and introns are distinguished in yellow boxes and in black lines, respectively.

FIGURE 3

Localization of cotton Rbohs on chromosomes. The genomic (At and Dt) analysis of Rboh genes in panels (A) G. barbadense and (B) G. hirsutum vis-à-vis the diploid Gossypium spp. Different-colored links indicate translocation from the A or D sub-genome of an allotetraploid cotton to the diploid cotton. Lined gene coupling represents a shared identity between them that exceeded 91%. The inter-genomic (At and Dt) analyses of Rboh genes in panels (C) G. barbadense and (D) G. hirsutum between the A and D sub-genomes. Different-colored lines indicate gene couples whose identity is over 98%, depicted using the Circos genome visualization tool (http://www.circos.ca/).

FIGURE 4

GbRbohs in the CladeD respond to V. dahliae infection and hormones in G. barbadense. Quantitative analyses of each clade GbRbohs’ expression levels in “Xinhai15” cotton plant leaves treated with (A) V. dahliae, (B) jasmonic acid, and (C) abscisic acid. Total RNAs were extracted from leaves of 3-week-old seedlings at 0, 4, 8, 12, and 24 h post-inoculation. The values are the mean ± SD for three technical replicates. Relative expression levels of all clade genes were determined after normalizing to the expression level at 0 h, which was set to 1.0. hpi, hours post-inoculation. The experiments were repeated at least three times, with similar results.

FIGURE 5

GbRboh5/18 are the main functional genes of CladeD in G. barbadense. Quantitative analyses of GbRboh5/18 expression levels in “Xinhai15” cotton plant leaves inoculated with (A) V. dahliae, (B) jasmonic acid, and (C) abscisic acid. Total RNAs were extracted from leaves of 3-week-old seedlings at 0, 4, 8, 12, and 24 h post-inoculation. The experiments were repeated three times, with similar results. The values are the means ± SD for three technical replicates. Transcript levels of each gene couple were first normalized to UBQ7. Asterisks indicate significant differences compared with 0 h under same treatment (*p < 0.05; **p < 0.01, Student’s t-test).

FIGURE 6

Knock-down of GbRboh5/18 attenuates plant resistance to V. dahliae. (A) Disease phenotype of cotton plants (“Xinhai15”) at 25 days post-infection with V. dahliae strain Vd991. Seedlings were inoculated with V. dahliae two weeks after VIGS and photographed 25 days later. (B) Expression of GbRboh5/18 in GbRboh5/18-VIGS and control plants. UBQ7 was the internal reference gene (**p < 0.01; Student’s t-tests). (C) Disease index of GbRboh5/18-VIGS and control plants at 21 and 25 dpi with V. dahliae. Asterisks indicate significant differences compared with TRV: 00 at same time (*p < 0.05; **p < 0.01, Student’s t-test). (D) Lesion area of GbRboh5/18-VIGS and control plants’ stems at 25 dpi with V. dahliae. Transverse section of stems were photographed with an OLYMPUS CX31 microscope. The values are the mean ± SD, n ≥ 30. Bar = 0.5 mm. (E) Fungal recovery assay. Sterile stems approximately 1 cm from same position of plants was plated on PDA plates at 25°C to allow V. dahliae recovery; photographs were taken 3 days post-inoculation. All experiments were repeated at least three times, with similar results.

FIGURE 7

GbRboh5/18 promotes ROS accumulation in G. barbadense. (A) Trypan blue staining of cell death and (B) DAB staining of ROS in GbRboh5/18-VIGS and control leaves. Leaves were stained at 48 h post-inoculation with or without V. dahliae. Leaves were pretreated with or without 10 μM DPI before trypan blue staining or DAB staining. Bar = 1 cm. (C) Schematic diagram showing the structure of overexpression vector. Gray-filled squares indicate ORF of GbRboh5/18; the left pentagon and right pentagon are the 35S promoter and 35S terminator, respectively. (D) Expression of GbRboh5/18 in GbRboh5/18-OE and vector plant leaves. UBQ7 was the internal reference gene. (*p < 0.05; Student’s t-tests). (E) H₂DCF-DA fluorescence probe of ROS in GbRboh5/18-OE and vector leaves cells. Bar = 20 μm. After 5 days of growth, the first true leaves were used to Q-PCR or H₂DCF-DA staining. All experiments were repeated at least three times with similar results.