Transcriptome profiling of Gossypium barbadense inoculated with Verticillium dahliae provides a resource for cotton improvement

Abstract

Background: Verticillium wilt, caused by the fungal pathogen Verticillium dahliae, is the most severe disease in cotton (Gossypium spp.), causing great lint losses worldwide. Disease management could be achieved in the field if genetically improved, resistant plants were used. However, the interaction between V. dahliae and cotton is a complicated process, and its molecular mechanism remains obscure. To understand better the defense response to this pathogen as a means for obtaining more tolerant cultivars, we monitored the transcriptome profiles of roots from resistant plants of G. barbadense cv. Pima90-53 that were challenged with V. dahliae.

Results: In all, 46,192 high-quality expressed sequence tags (ESTs) were generated from a full-length cDNA library of G. barbadense. They were clustered and assembled into 23126 unigenes that comprised 2661 contigs and 20465 singletons. Those unigenes were assigned Gene Ontology terms and mapped to 289 KEGG pathways. A total of 3027 unigenes were found to be homologous to known defense-related genes in other plants. They were assigned to the functional classification of plant-pathogen interactions, including disease defenses and signal transduction. The branch of "SA→NPR1→TGA→PR-1→Disease resistance" was first discovered in the interaction of cotton-V. dahliae, indicating that this wilt process includes both biotrophic and necrotrophic stages. In all, 4936 genes coding for putative transcription factors (TF) were identified in our library. The most abundant TF family was the NAC group (527), followed by G2-like (440), MYB (372), BHLH (331), bZIP (271) ERF, C3H, and WRKY. We also analyzed the expression of genes involved in pathogen-associated molecular pattern (PAMP) recognition, the activation of effector-triggered immunity, TFs, and hormone biosynthesis, as well as genes that are pathogenesis-related, or have roles in signaling/regulatory functions and cell wall modification. Their differential expression patterns were compared among mock-/inoculated- and resistant/susceptible cotton. Our results suggest that the cotton defense response has significant transcriptional complexity and that large accumulations of defense-related transcripts may contribute to V. dahliae resistance in cotton. Therefore, these data provide a resource for cotton improvement through molecular breeding approaches.

Conclusions: This study generated a substantial amount of cotton transcript sequences that are related to defense responses against V. dahliae. These genomics resources and knowledge of important related genes contribute to our understanding of host-pathogen interactions and the defense mechanisms utilized by G. barbadense, a non-model plant system. These tools can be applied in establishing a modern breeding program that uses marker-assisted selections and oligonucleotide arrays to identify candidate genes that can be linked to valuable agronomic traits in cotton, including disease resistance.

Figure 1

Infection of cotton seedlings with Verticillium dahliae. A: Aseptic growth in tissue culture flask. B: status at 3 dpi for roots treated with conidial suspension. C: Infection process, based on GFP-tagged V. dahliae strain. D: Vascular tissue of uninfected hypocotyl section (CK). Severe browning of vascular tissue in longitudinal section at 7 dpi. E: Mycelia growth in vascular tissue of surface-sterilized hypocotyl section prepared from cotton seedling at 2 dpi, then incubated for 3 d on 25% potato dextrose agar. F: Mycelium observed with optic microscope.

Figure 2

Detection and analysis of inserts for cDNA clones. PCR amplification of inserts for cDNA clones (A) and statistical analysis of insert fragment sizes in cDNA library (B). Lanes: M, DL5000 marker; 1-22, random clones from library.

Figure 3

Distribution of 1981 contigs based on number of clustered ESTs.

Figure 4

Functional classification of unigenes from Verticillium dahliae-stressed cotton within categories of biological processes, molecular functions, and cellular components.

Figure 5

Classifications for Clusters of Orthologous Groups (COGs). Sequences with Nr hits were grouped into 22 COG classes.

Figure 6

Example of KEGG pathways found for full-length cDNA clone ESTs. Each box shows enzymes involved in each section of pathway. Genes highlighted in red were detected from our full-length cDNA library.

Figure 7

Comparisons of 23,126 unigenes from cotton and other plant species. Searches were performed against nucleotide databases for ESTs (A) or proteins (B) using Blastn or Blastx (E-value ≤10^-5).

Figure 8

Detailed expression profiles of defense-related genes. Q-PCR analysis was conducted for transcription levels of selected genes in response to V. dahliae infection in mock-inoculated and fungal-inoculated roots at 1, 2, 4, 6, 8, 12, 24, 36, 48, 72, 96, and 120 hpi. Data within each column are means and standard errors (bar) for 3 independent Q-PCR experiments using 3 technical replicates; vertical bars indicate standard errors. Transcription level is represented as ratio of Ct value for studied gene, calibrated to mock-inoculated control and normalized to Ct value for GhUBQ14 and cotton actin.