A CC-NBS-LRR gene induces hybrid lethality in cotton

Abstract

Hybrid lethality forms a reproductive barrier that has been found in many eukaryotes. Most cases follow the Bateson-Dobzhansky-Muller genetic incompatibility model and involve two or more loci. In this study, we demonstrate that a coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR) gene is the causal gene underlying the Le4 locus for interspecific hybrid lethality between Gossypium barbadense and G. hirsutum (cotton). Silencing this CC-NBS-LRR gene can restore F1 plants from a lethal to a normal phenotype. A total of 11 099 genes were differentially expressed between the leaves of normal and lethal F1 plants, of which genes related to autoimmune responses were highly enriched. Genes related to ATP-binding and ATPase were up-regulated before the lethal syndrome appeared; this may result in the conversion of Le4 into an active state and hence trigger immune signals in the absence of biotic/abiotic stress. We discuss our results in relation to the evolution and domestication of Sea Island cottons and the molecular mechanisms of hybrid lethality associated with autoimmune responses. Our findings provide new insights into reproductive isolation and may benefit cotton breeding.

Keywords: ATP-binding; DNA-dependent ATPase activity; NBS-LRR genes; autoimmunity response; cotton; hybrid lethality; map-based cloning.

Fig. 1.

Phenotypic characterization of cotton hybrid lethality. (A) Phenotypes and genotypes of the normal parent lines TM-1 and Coastland R4-4 in the field in Nanjing, and of lethal F₁ plants in the field in Nanjing and Sanya. The blue arrow indicates necrotic spots and yellow arrows indicate flower buds. D11 and D8 refer to chromosomes D11 and D08, respectively; (B) Leaves from lethal and normal F₁ plants at four different developmental stages (S1–S4). The scale bars in images of complete leaves are 1 cm; in images of leaf details the bars are 0.1 cm (C) Chlorophyll content of leaves of normal and lethal F₁ plants. (D) TEM images of leaves of lethal and normal F₁ plants in stage S4. CP, chloroplast; CW, cell wall; SG, starch grain. Chloroplast envelopes of lethal F₁ plants are blurred and the thylakoid membrane is abnormal.

Fig. 2.

Map-based isolation of the cotton hybrid lethality gene Le₄. (A) Le₄ was mapped on the D11 chromosome between the markers BNL3279 and BNL1154 using an F₂ population. The genetic distance between the markers was 9.4 cM. Le₄ was further fine-mapped to a region between markers W8424 and K1805 using 734 dominant individuals. A 267-kb region containing three putative ORFs was obtained by mapping this region with sequences from G. hirsutum TM-1. (B) Transcriptome analysis was used to compare the expression levels of Le₄ in leaves from lethal F₁ and normal F₁ plants at four different developmental stages (S1–S4). Data are means of two or three replicates. (C) qRT-PCR analysis was used to compare the transcript levels of Le₄ in leaves from lethal F₁ and normal F₁ plants at four different developmental stages. Data are means (±SE) of three replicates. Significant differences between means were determined using Student’s t-test: **P<0.01.

Fig. 3.

Phenotypes of virus-induced gene silencing (VIGS) plants and suppression of endogenous transcripts in cotton. (A) Phenotypes of lethal F₁ (TM-1×R4-4) plants. Detailed images of the leaves are at 25× magnification. (B) qRT-PCR analysis was used to examine the transcript levels of Le₄ in leaves from plants infected with pTRV2 or pTRV2- Le₄. Data are means (±SE) of three replicates. Significant differences between means were determined using Student’s t-test: **P<0.01. (C) TEM images of leaves of lethal and VIGS-silenced F₁ plants.

Fig. 4.

DNA dependent ATPase activity and ATP binding genes were highly expressed during developmental stages S1 and S2 in lethal F₁ plants compared with normal F₁ plants. (A) Enriched molecular function GO terms in up-regulated genes in the lethal F₁ plants at S1 and S2. (B) Heatmap of the fold-change in expression of DNA-dependent ATPase and ATP-binding genes in lethal F₁ plants. Yellow/brown indicates genes up-regulated in lethal F₁ plants, blue/grey indicates genes down-regulated in lethal F₁ plants, and white indicates genes with no difference in expression between lethal and normal F₁ plants. (C) qPCR confirmation of the six differentially expressed ATP binding genes. Data are means (±SE) of three replicates. Significant differences between means were determined using Student’s t-test: **P<0.01.

Fig. 5.

A proposed model of hybrid lethality between G. barbadense and G. hirsutum. Normally, Le₄ can cycle through ON and OFF states, and favors the OFF state. When it is bound to ATP, Le₄ is activated and changes from OFF to ON. Le₄ interacts with Le₃ directly or indirectly and triggers an immune response. Immune signals transmitted by reactive oxygen species (ROS) crosstalk and MAPK signaling pathways integrate with defense and growth hormone signals, leading to crosstalk and amplification of downstream genes. Constitutively activated defense responses result in the suppression of plant growth and ultimately induce plant death.