Functional Genomics

Mitogen-activated protein kinase (MAPK) cascades are evolutionarily conserved signal transduction modules that are found in all eukaryotes. MAPKKKs (MAPK kinase kinases) are important components of the MAPK cascades, operate at the top levels of these cascades and have the most family members. This gene family has been investigated in Arabidopsis, rice and maize, but here has been no systematic investigation in cotton. Recently, the Gossypium raimondii genome was sequenced, which made it possible to identify all the MAPKKKs in this species. In this study, 78 MAPKKK genes were identified in G. raimondii by sequence comparison and signature motif searches. Most of MAPKKK proteins were predicted to be located in the nucleus and the cytoplasm. Based on phylogenetic analysis, MAPKKKs were classified into three subfamilies, of which 12 were ZIK genes, 22 were MEKK genes and 44 were Raf genes. The numbers and the conserved protein motifs within the subfamilies were similar to Arabidopsis and rice. The ZIK and MEKK genes displayed a scattered genomic distribution across 11 of the 13 chromosomes, whereas Raf genes were distributed across the entire genome. Gene structure analysis identified some losses or gains of exons during the evolution of these family genes. Their conserved patterns observed for introns and additional domains were consistent with the evolutionary relationships inferred from the phylogenetic analysis within subfamily. Furthermore, the expression pattern of MAPKKKs was analyzed in mature leaves and at the early fiber cell development stage. Sixty MAPKKK genes were expressed, of which 41 were strongly expressed in mature leaves. Twelve MAPKKK genes were more highly expressed in 3 days post-anthesis (DPA) ovules than in 0-DPA ovules. The tissue-specific expression patterns suggest their important roles in different tissues and at the developmental stage. Our results provide a foundation for future evolutionary and functional characterizations of MAPKKK genes in cotton and probably other Gossypium plants.

Verticillium wilt is the most important fungal disease that causes severe losses in yield and fiber quality of cotton in many cotton-growing areas. Developing tolerant cotton cultivars by incorporating genes from resistant germplasm is now regarded as a potential strategy for controlling this disease. Here, we identified a gene of hybrid proline-rich protein in Gossypium barbadense, designated as GbHyPRP1, which encodes a protein containing a proline-rich repetitive domain (PRD) at the N-terminus and a hydrophobic C-terminus Pollen Ole e I domain that is mostly expressed in the root and stem. When the cotton seedlings were inoculated with Verticillium dahliae, the expressional level of HyPRP1 dropped drastically within 48h of monitoring in roots, whereas in stems the transcript was kept unchanged. To better characterize the function of GbHyPRP1 in cotton resistance to V. dahliae, we employed virus-induced gene silencing (VIGS) to suppress endogenous HyPRP1 in G. barbadense. As a result, HyPRP1-silenced plants displayed more resistant to V. dahliae, with a dramatic increase in length of primary roots compared to the control. Additionally, we transformed GbHyPRP1 into V. dahliae-susceptible Arabidopsis ecotype Columbia by Agrobacterium-mediated transformation. Statistical analysis of the disease index showed that overexpression of GbHyPRP1 compromised transgenic Arabidopsis plants resistance to V. dahliae. Furthermore, a GbHyPRP1 promoter region of 1431 bp was isolated by PCR and has been validated by transgenic Arabidopsis carrying the GUS ( -glucuronidase) reporter gene fused to the 1431 bp fragment, and T2 plants were used for GUS histochemical assay. Putative cis-acting elements in the promoter were sought in PLACE database. Several distinct cis-acting regulatory DNA elements associated with the response to abiotic/biotic stresses and phytohormone were identified, such as MYB recognition site, ABRE (ABA-responsive element), ERE (ethylene-responsive element) etc. Especially, we further verified cis-acting element of ABRE through the response of GbHyPRP1 transcripts to exogenous ABA in cotton plants.The result showed that GbHyPRP1 was markedly down-regulated after 6-24 h of ABA treatment. Overall, our results suggest that GbHyPRP1 performs a role in negative regulation of the cotton resistance to V. dahlia, and has a potential value for the resistance improvement of cotton Verticillium wilt.

Verticillium wilt, caused by Verticillium dahliae, brings serious damage to cotton production. Gossypium barbadense is one of the most important resistance sources. The knowledge of mechanism underlying its resistance to Verticillium wilt is still limited. To get an overview of transcriptome characteristic in roots of Gossypium barbadense resistant cultivar Pima90-53 compared to G. hirsutum susceptible cultivar CCRI8 during infection by V. dahliae, a high-throughput RNA sequencing based on next generation sequencing (NGS) were performed. Totally, 83,964 unigenes were generated and assigned to known protein databases including NCBI non-redundant protein database (nr) (43,248, 51.51%), Swiss-Prot (27,414, 32.65%), Clusters of orthologous groups (COG) (16,723, 19.92%) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) (10,437, 12.4%), as determined by Blastx search. With another NGS based platform, the method described by Audic et al. was applied to identify differentially expressed genes from the normalized DGE data by pair-wise comparisons between the “Pima90-53”and “CCRI8” at five infection stages (6, 12, 24, 36 and 48 hpi), with the aim of identifying genes involved in V. dahliae resistance. Differentially regulated cotton genes (2-fold or greater change; P≤0.001) upon inoculation with the V. dahliae were identified at different time points when compared with mock-inoculated control plants. V. dahliae inoculation resulted in that most of defense responses in Pima90-53 were more rapidly induced than those in CCRI8. And the difference in response intensity between the resistant and susceptible cultivars is also reflected by the number of induced genes, which amounted to 2628 genes at 36 hpi and 6124 genes at 48 hpi in Pima90-53 and CIR8, respectively. In addition, V. dahliae inoculation resulted in repression of photosynthesis. To characterize the functional consequences of gene expression changes associated with infection with V. dahliae in the resistant cultivar, pathway analysis of differentially expressed genes was performed, based on the KEGG database using the two-side Fisher’s exact test. Several metabolic pathways that are related to immunity were selected for further analysis. The selected pathways included Perception of PAMPs by Pattern Recognition Receptors, Effector-triggered immunity, Ion fluxes, Transcription factors, Oxidative burst, Pathogenesis-related proteins, Programmed cell death, Plant hormones and Cell wall modification. All differentially expressed unigenes are involved in their cognition of PAMPs, and the high accumulated levels of defense-related transcripts may contribute to V. dahliae resistance in cotton. Based on the transcriptomic comparison of two differentially expressed unigenes between “Pima90-53”and “CCIR8”, a schematic illustration of plant defense in G. barbadense against V. dahliae was constructed. In all, this study generated a substantial amount of cotton transcript sequences and compared the defense responses against V. dahliae between resistant and susceptible cottons. The results contribute to the identification of candidate genes related to plant resistance in a non-model organism, cotton, and help to improve the current understanding of host-pathogen interactions.

Lignin metabolism has a central role in the cotton resistance to wilt fungus V. dahliae. Here, we cloned a gene, GhLaccase (G. hirsutum laccase, EU642559.1), from the Verticillium-wilt-tolerant cotton. Bioinformatics analysis showed that a cleaved signal sequence, MGLQQGFVTWFVGVLFLTTLLLSSA, existed in N-terminal of GhLaccase protein. The GhLaccase protein was located in cell wall, suggesting that GhLaccase performed its active in extracellar. The expression of GhLaccase in tolerant cotton cv. Jimian20 increased 8-14 fold compared to that of control and peaked in 8 hr post inoculation with V. dahliae. Silencing of GhLaccase in the cotton by VIGS method declined the Verticillium wilt tolerance, and over-expression of GhLaccase enhanced the resistance in Arabidopsis. The contents of Klason lignin, total lignin and guaiacyl monomers (G monomers) in transgenic Arabidopsis over-expression lines were highly significant higher (P<0.01) than those of wild type inoculated with V. dahliae or not. Our results showed that GhLaccase from G. hirsutum was involved in lignin content alteration and Verticillium wilt resistance.

Verticillium dahliae is a destructive, soil-borne fungal pathogen that causes vascular wilt disease in many economically important crops worldwide. Polyamine (PA) is ubiquitous aliphatic amine that has been implicated in various physiological and developmental processes in all living organisms. Plant polyamine oxidase (PAO) involved in the terminal catabolism of PA has been previously hypothesized down-regulating of PA level with enhanced plant defense against various pathogens. However, the effect of engineering PAO in host plants is rarely known on the hemibiotrophic fungal pathogen V. dahliae, and the underlying mechanism of PA on defense response in plants is poorly understood. Here, a novel polyamine oxidase (GhPAO) was identified by screening SSH library of G. hirsutum cv. ‘Jimian 20’ and the cDNA library of G. barbadense cv. ‘Pima 90-53’. Its expression was rapidly induced in the resistant ‘Jimian 20’ and suppressed in the susceptible ‘Han 208’ after V. dahliae inoculation. Overexpression of GhPAO in Arabidopsis thaliana exhibited improved resistance to V. dahliae, and unexpectedly elevated all of three PA, putrescine, spermidine and Spm, by driving ten genes accounting for PA biosynthesis (AtADC1/2, AtSAMDC, AtSPDS1/2, AtSPMS and AtPAO1/2/3/4/5) after V. dahliae infection with real-time RT-PCR analysis. The level of hydrogen peroxide (H2O2), as the product of PAO-derived PA oxidation and the signaling component, was elevated pre- and post-inoculation in GhPAO overexpression lines when compared to the wild type with ELISA analysis. The hormone salicylic acid and phytoalexin camalexin were distinctly increased in GhPAO overexpression lines after V. dahliae inoculation when compared to the wild type by HPLC experiment. PA and H2O2 showed efficient inhibition to V. dahliae conidial proliferation and hyphal growth via dual culture technique in vitro, and Spm exhibited the highest antifungal activity. Moreover, Spm and H2O2 could promote phytoalexin camalexin biosynthesis via inducing three pathogen-responsive mitogen-activated protein kinases (MPKK1, MPK3 and MPK6) and two downstream Cytochrome P450 proteins (CYP71A13 and PAD3), with exogenous application of Spm and H2O2 in the wild type pre-inoculation. In addition, quantification of free PA by HPLC in cotton showed that all of three PA were gradually decreased in two susceptible upland cotton, ‘Han 208’ and ‘CCRI 8’, after V. dahliae inoculation, while showed increase in two resistant sea island cotton, ‘Pima 90-53’ and ‘Hai 7124’. In all, GhPAO, a novel PAO from cotton, contributes to plant resistance against the hemibiotrophic fungal pathogen V. dahliae via elevating secondary antimicrobial compounds, PA, H2O2 and phytoalexin camalexin, and plant hormone salicylic acid levels.

5-enolpyruvoylshikimate-3-phosphate synthase(EPSPS), a key enzyme exists in the shikimate pathway of aromatic amino acid biosynthesis in nearly all plants, bacteria, and fungi, is also the target enzyme of the herbicide glyphosate (N-(phosphonomethyl)glycine). Cloning of EPSPS gene could provide candidate gene for creating glyphosate-resistant genetically modified crops. One EPSPS gene has been cloned from upland cotton(Gossypium hirsutum) and its complete CDS has been submitted to NCBI(Accession number: EU797516). The completed genome sequencing work of a diploid cotton Gossypium raimondii makes it possible to analysis cotton EPSPS gene in genomic level. A highly homologous gene of this cotton EPSPS was identified based on a blast assay using the EPSPS complete CDS in the genome database of Gossypium raimondii of JGI(http://www.phytozome.org). In order to facilitate the distinction of this two genes, the EPSPS gene was named EPSPS1 and its homologous gene was named EPSPS2. EPSPS1 is located at eighth chromosome with locus name Gorai.008G113600, while EPSPS2 is located at first chromosome with locus name Gorai.001G174400. These two genes in genome are in opposite directions. The length of coding region sequence of EPSPS1 and EPSPS2 are 3080 bp and 3341 bp respectively, but the CDS length of these two genes is same as 1566 bp. Both of them have the same number of exons, and the same length of corresponding exons. This shows that the introns disparity caused the main difference between these two genes. The similarity index of coding region sequence, CDS and translated amino acid sequence of these two genes are 44.2%, 86.2% and 89.2% respectively. The 74 N-terminal amino acid residues of these two amino acid sequences is chloroplast transit peptide, both of which doesn’t have homology. This research provides a theoretical basis for cloning a homologous gene of cotton EPSPS.

Natural colored cotton is an important cotton germplasm and potential dye-free textile material. However, the study involved the molecular mechanism of fiber development and pigmentation formation are very less. RT-white and Brown1-61, the near-isogenic lines of upland cotton with white and brown fibres, were analyzed. We found that immature fibres of white and brown cotton at 28 days post-anthesis (dpa) contained flavones, whereas brown cotton fibres at 13dpa, 17dpa, 23dpa, leaf, flower, and bud contained flavonols. But Brown1-61 mature fibres may contain procyanidin (tannin).Using the RNA-seq technology which provides a powerful tool to reveal the whole transcriptome profiling of developmental colored fiber, the most important pigment biosynthesis pathways in plant, including &quot;flavonoid biosynthesis&quot; and &quot;anthocyanins biosynthesis&quot; pathways were analyzed. The result demonstrated that the pigmentation formation in brown color cotton fiber was possibly the consequence of interaction of oxidized tannins and glycosylated anthocyanins.

Cotton is a good model for studying polyploidy effects on gene expression and fiber trait domestication. The most widely cultivated Upland cotton is an allotetraploid derived from A- and D- progenitor species, which diverged 10-12 million years ago. A-genome diploid progenitors produce spinnable fibers, while D-genome diploid progenitors bear very short and poor fibers. The superior fiber trait in the allotetraploid cotton is domesticated, which is subjected to changes in the expression of homoeologouse genes. One pair of the homoeologous genes, encoding the MYB-domain transcription factor MYB2, promotes fiber development in cotton and is functionally homologous to Arabidopsis glabrous1 (GL1) in trichome formation. MYB2A and MYB2D homoeologs in the allotetraploid cotton (AADD) are derived from A-genome and D-genome species, respectively. Overexpressing MYB2A but not MYB2D complemented the gl1 phenotype. This functional divergence of MYB2 homoeologs is associated with miR828 and miR858 preferentially targeting and the generation of trans-acting siRNAs (ta-siRNAs). Mutating the miR828-binding site or replacing its downstream sequence in MYB2D abolished ta-siRNA production and restored trichome development. Single nucleotide polymorphism (SNP) that is adjacent to the 3’ end of miRNA targeting site on MYB2 genes affects the homoeologs functional diversity by altering the miRNA accessibility and protein sequence. These data support a unique role for miRNAs in functional diversification of target homoeologous genes that are important to evolution and selection of morphological traits.

Verticillium wilt causes massive yield losses of cotton, but the mechanism of cotton resistance to Verticillium dahliae is complex and poorly understood. Proteomic and transcriptome are efficient measures to explore disease resistance response genes in cotton infected with V. dahliae. Combined with virus induced gene silencing (VIGS), it is possible to uncover the molecular mechanism of cotton to V. dahliae quickly. Comparative proteomic analysis was performed in Gossypium barbadense cv. '7124' upon infection with V. dahliae. A total of 188 differentially expressed proteins were identified by mass spectrometry (MALDI-TOF/TOF) analysis and could be classified into 17 biological processes based on Gene Ontology annotation. Most of these proteins were implicated in stimulus response, cellular processes and metabolic processes. Based on the proteomic analysis, several genes involved in secondary metabolism, reactive oxygen burst and phytohormone signaling pathways were identified for further physiological and molecular analysis. Based on the results, we suggest that the production of gossypol is sufficient to affect the cotton resistance to V. dahliae. Silencing of GbCAD1, a key enzyme involving in gossypol biosynthesis, through VIGS compromised cotton resistance to V. dahliae. Reactive oxygen species and salicylic acid (SA) signaling may be also implicated as regulators in cotton responsive to V. dahliae according to the analysis of GbSSI2, an important regulator in the crosstalk between SA and jasmonic acid (JA) signal pathways. Moreover, brassinosteroids (BRs) and JA signaling may play essential roles in the cotton disease resistance to V. dahliae. The BR signaling was activated in cotton upon inoculation with V. dahliae and the disease resistance of cotton was enhanced after exogenous application of brassinolide (BL). Meanwhile, JA signaling was also activated in cotton after inoculation with V. dahliae and BL application. HDTF1 encoded putatively a nuclear homeodomain transcription factor and was suppressed in cotton upon inoculation with V. dahliae and Botrytis cinerea. Silencing of HDTF1 significantly enhanced cotton resistance to V. dahliae and B. cinerea. Meanwhile, accumulation of the phytohormone jasmonic acid (JA) and activation of JA-related signal pathway were found in HDTF1-knockdown plants. While, no difference was found in content of salicylic acid (SA) and the expression of SA-related genes between the control and HDTF1-silenced cotton. Based on the studies, HDTF1 seems to be an important factor in regulating JA signaling and disease resistance to V. dahliae and B. cinerea in cotton. All these results provide highlights in the molecular basis of cotton resistance to V. dahliae.

From cotton genomics to functionomics via Agrobacterium-Mediated VIGS Assay Libo Shan Department of Plant Pathology and Microbiology Institute of Plant Genomics and Biotechnology Texas A&M University lshan@tamu.edu As an economically important crop, cotton (Gossypium spp.) serves as a significant source of textile fiber, feed and oil products worldwide. Cotton production is hindered by various biotic and abiotic stresses. The genetic and molecular mechanisms mediating cotton stress responses remain poorly understood. We have previously developed an Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in several cotton cultivars. The genes of interest could be potently and readily silenced at the seedling stage with a consistently high efficiency. To monitor gene silencing efficiency, we have cloned cotton GrCla1 from G. raimondii, a homolog gene of Arabidopsis Cloroplastos alterados 1 (AtCla1) involved in chloroplast development. Silencing of GrCla1 results in albino phenotype on the newly emerging leaves, serving as a visual marker for silencing efficiency. To further facilitate cotton functional genomic studies and identify cotton genes mediating biotic and abiotic stress responses, we constructed a VIGS library from diploid cotton G. raimondii. Sequencing of the representative colonies revealed that the library covers a significant percentage of cotton unique genes with about 50X coverage of predicted cotton protein-coding genes. As a proof-of-concept experiment, we screened the genetic determinants involved in cotton drought tolerance using this VIGS library. We further established a cotton protoplast system to study gene functions by a gain-of-function approach. These technique advances made it possible to dissect the molecular and biochemical regulatory mechanisms of cotton genes under different biotic and abiotic stresses.