Functional Genomics

For ICGI 2018. Cotton fiber is the most important sustainable fiber source for textile industry. It is a single cell organ derived from the epidermis of the cotton ovule or seed. To understand the molecular basis of plant cell differentiation pattern, the cotton fiber cell is a good model system. Non-coding RNA is emerging as one of the most important regulators for the gene expression in response to multiple biological transitions and environmental stimuli. We systematically investigate the non-coding RNA behavior in the fiber cell differentiation progress. The major data indicate both small RNA and long non-coding RNA (lncRNA) play critical roles in fiber cell development. First of all, the fiber cell generates a unique group of small RNA in the fiber initiation stage. For example, the miR828 and miR858 trigger the target gene GhMYB2 to generate tasiRNAs in fiber cell fate determination. Another MIXTA MYB transcriptional factor coding gene, GhMML3 can generate an antisense transcript on the 3’ end of the gene loci. Together with the sense and antisense transcripts, a double strand RNA come into being to derive small RNAs. These secondary generated small RNAs interfere the cell fate determination in the mml3 mutant in stimulating the fiberless seed phenotype. On the other hand, the long non-coding RNAs are also found to take parts in the fiber cell differentiation by small RNA generation. We therefore conclude noncoding RNAs directly impact the fiber cell development in multiple aspects of molecular regulation.

Plants evolve effective mechanisms to protect themselves against multiple stresses and employ jasmonates (JA) as vital defense signals to defend against pathogen infection. Accumulation of JA induced by signals from biotic and abiotic stresses, results in degradation of Jasmonate-ZIM-Domain (JAZ) proteins, and then de-repressed the JAZ-repressed transcription factors (such as MYC2) to activate defense responses and developmental processes. Here, we characterized a JAZ family protein, GhJAZ2, from cotton (Gossypium hirsutum) which was induced by methyl jasmonate (MeJA) and inoculation of Verticillium dahliae. Over-expression of GhJAZ2 in cotton impairs the sensitivity to JA, decreases the expression level of JA-response genes (GhPDF1.2 and GhVSP) and enhances more susceptibility to V. dahliae and insect herbivory. Yeast two-hybrid and BiFC assays showed that GhJAZ2 may be involved in regulation of cotton disease resistance by interacting with more disease response proteins, like pathogenesis-related protein GhPR10, dirigent-like protein GhD2, NBS-LRR disease resistant protein GhR1, and a basic helix-loop-helix transcription factor bHLH171. Unlike MYC2, over-expression of bHLH171 in cotton activates the JA synthesis and signaling pathway, and improves plant tolerance to fungus V. dahliae. Molecular and genetic evidences showed that GhJAZ2 could interact with bHLH171 and inhibit its transcriptional activity, as a result, restrain the JA-mediated defense response. Recent studies have revealed that the SUPPRESSOR OF BIR1-1 (SOBIR1) can interact with multiple receptor-like proteins (RLPs) and is required for resistance against fungal pathogens. We also find that GbSOBIR1 gene could be induced by Verticillium dahliae inoculation. Knock-down of GbSOBIR1 resulted in attenuated resistance of cotton plants to V. dahliae, while heterologous overexpression of GbSOBIR1 in Arabidopsis improves the resistance. We also found that the kinase region of GbSOBIR1 could interact with bHLH171 and contributes to the resistance of cotton against V. dahliae. And the transcriptional activity of bHLH171 is significantly improved when co-expressed with GbSOBIR1 in tobacco. To identify the GbSOBIR1-mediated phosphorylation site of bHLH171, a spectrometric analysis was performed, and phosphorylation defective forms of the bHLH171 protein with serine to alanine mutations were assayed. The results showed that phosphorylation at Ser413 is essential for the physiological function of bHLH171. Collectively, these results demonstrate that multiple signal pathways are employed by cotton during cotton resistance to V. dahliae.

GhAPX1AT/DT (Ascorbate peroxidase) encoded one member of the previously unrealized group of cytosolic APXs (cAPXs) that were preferentially expressed during the fiber elongation stage. Suppression of all cAPX (IAO) resulted in a 3.5-fold increase in H2O2 level in fiber and oxidative stress, and significantly suppressed fiber elongation. The fiber of over-expression lines exhibited higher tolerance to oxidative stress. Differentially expressed genes (DEGs, by RNA-seq) in 10 DPA fiber of IAO lines were related to redox homeostasis, signaling pathways, stress responses and cell wall synthesis, and the DEGs that were up-regulated in IAO lines were also up-regulated in the 10 DPA and 20 DPA wild cotton fiber compared with domesticated cotton. These results suggest that optimal redox state regulated by cytosolic APX are key mechanisms regulating fiber elongation. Furthermore, suppression of cAPX increased ASC contents and delayed tissue browning by maintaining ferric reduction activity under Fe-deficient conditions in the ovule culture system. Meanwhile, APX RNAi line also exhibited the activation of expression of iron-regulated transporter (IRT1) and ferric reductase–oxidase2 (FRO2) to adapt to Fe deficiency.

DNA methylation regulates gene expression without changing the original DNA sequence, which regulates a range of functions in plant development and stress responses, maintenance genomic stability, stress response, and among others, but a role in male sterility under HT remains undetermined. In our previous studies, male sterility under high temperature is a critical factor contributing to yield loss in cotton. Using genome-wide total DNA methylation rate measurement by HPLC at three anther developmental stages under normal and high temperature (HT) conditions, we found that the total DNA methylation level of H05 was always lower than that in 84021 under HT, which was in accordance with more differentially expressed genes in H05 than in 84021 under HT (Min et al., 2014). To better understand how DNA methylation addresses HT stress during male reproductive stages, we performed whole genome bisulfite sequencing. Global disruption of DNA methylation, especially CHH methylation (where H=A, C or T), was found in an HT-sensitive line. Changes of 24-nucleotide small interference RNAs were significantly associated with DNA methylation levels. Experimental suppression of DNA methylation led to pollen sterility in the HT-sensitive line under NT, but did not affect the normal dehiscence of anther wall. Further transcriptome analysis of the anther showed that the expression of genes in sugar and reactive oxygen species (ROS) metabolic pathways were modulated significantly, but auxin biosynthesis and signaling pathways were slightly changed, indicating that HT disorders sugar and ROS metabolism via disrupting DNA methylation, leading to microspores sterility. This study opens up a path to create HT-tolerant cultivars using epigenetic solutions.

It is generally believed that the ancestors of Gossypium arboreum (A2 genome) or G. herbaceum (A1 genome) together with that of G. raimondii (D5 genome) provided the genetic basis for the modern, cultivated allotetraploid cotton species G. hirsutum (AtDt genome). Here we upgraded the A2 genome assembly by integrating SMRT sequencing and Hi-C technologies. We further resequenced 230 G. arboreum and 13 G. herbaceum accessions (average 6x depth) in China to generate a map of genome variation including ~18 million SNPs and ~2 million indels. Independent analysis suggested that Chinese G. arboreum originated in South China and was subsequently introduced to the Yangtze River and Yellow River regions. GWAS for 11 agronomically-important traits in G. arboreum identified a total of 98 significant peak associations. We found that a burst of transposable elements (TEs) contributed significantly to the 2-fold increase in the size of the G. arboreum genome, when compared to that of the D genome. Comparative genomics analysis suggested that an Ile/Val substitution in conserved catalytic motif DDVAE of 4 gossypol biosynthesis CDN1 genes renders the ability to produce gossypols only in cotton plants. Sequence alignments of the promoters of ACO gene, which is key to ethylene biosynthesis in cotton, revealed that specific deletions in both G. arboreum and in A-subgenome of G. hirsutum caused the loss of several cis-elements, including MYB binding sites, and may be responsible for the inactivation of ACO gene transcription in G. arboreum ovules. This reduced ACO gene expression correlated to the short-fibered phenotype in G. arboreum, whereas very high levels of ACO transcriptions, with a resultant ethylene burst, in G. raimondii ovules seems to force an early fiber senescence phenotype. A Cysteine/Arginine substitution of GaKASIII seems to have conferred significant alteration of the fatty acid composition (C16:0 and C16:1) in cotton seed. Also, gain of Fusarium wilt disease resistance in YZ and YR accessions is associated with GaGSTF9, whose expression is highly inducible upon fungal inoculation. The GaGSTF9-silenced cotton line shows sensitivity to fungal inoculation, whereas overexpression of cotton GaGSTF9 in A. thaliana displays resistance to Fusarium wilt disease compared to wild-type.

Verticillium wilt, caused by soil-borne fungus Verticillium dahliae, is one of the most destructive diseases in cotton. An LRR-TM gene we cloned from Gossypium barbadense (GbVe) significantly improved Verticillium wilt resistance of transgenic Arabidopsis. However, the resistance mechanism of the GbVe is still obscure. In the present study, we identified that the GbVe located in Dt09, homologous to tomato Ve1 but possessing lower identity with other reported cotton Ve genes in At01 and /or Dt01, and further investigated the molecular mechanism of the gene in resistance against V. dahliae. Ectopic expression of GbVe activated salicylic acid signaling, elevating the expression levels of EDS1, NDR1, PR4 and PR5 in transgenic Arobidopsis plants, and enhanced the accumulation of lignin, which improved resistance to V. dahliae. These results provide us important clue to further investigate the molecular mechanism of GbVe in cotton itself. Overexpression of GbVe in cotton (G. hirsutum) made defense response to Verticillium wilt enhanced. Transgenic cotton led to activation of SA signaling pathway (including cotton ICS1, EDS1, NPR1, NDR1, PR1, PR4 and PR5 genes), causing SA acumulation and lignin increase that confer resistance to V. dahliae. We also detected that the GbVe could interact with NHL13 gene involved in SA signal pathway via a yeast-two-hybrid screening. In the condition of V. dahliae free, GbVe and NHL13 interacted with each other. However, once stressed by the pathogen, the expression of NHL13 gene decreased whereas the expression level of GbVe displayed significantly increased, indicating that the interactions dissociation between GbVe and NHL13. The enhanced GbVe expression further activated defensive genes expression in SA signal pathway as well as regulating H2O2 and lignin content, which improved the Verticillium wilt resistance of cotton. When silenced GbVe gene in cotton, the plants showed more susceptible. Whereas, the plants displayed no obvious disease resistance difference between NHL13-silent and non-silent seedlings. These funding provides insight into molecular mechanism by which plants integrate SA signal regulated by GbVe to protect themselves from V. dahliae infection.

MAPK（mitogen-activated protein kinase）cascade plays important roles in responding to abiotic stresses for plants. In our present study, 23 association loci associated with seven drought-related traits were obtained based on GWAS（Genome-wide association study）method, and 50 differential expressed genes were selected as candidate ones combined with RNA-Seq data, among which one gene had a significant association with plant height trait under drought condition, named GhDRP1(Drought Response Protein1). GhDRP1 encodes a MPK (mitogen protein kinase) and has higher identity with Arabidopsis AtMPK4, then we created the overexpression (OE) and silencing (SI) GhDRP1 cotton materials, and found that OE plants were sensitive to drought stress, but resistant to drought treatment when expression was down-regulated. Yeast-two hybrid experiments found that GhDRP1 directly interacts with GhHT1 (High leaf temperature 1), and GhHT1 interacts with GhOST1 (Stomatal opening factor 1) which is a key factor regulating stomatal conductance, indicating that GhDRP1 plays key roles during cotton adaptation to drought stress. This study will broaden the knowledge of molecular mechanisms of cotton drought stress response, especially the theoretical basis for MAPK cascade regulating drought resistance in cotton, simultaneously providing important bridge parents for cotton drought resistance breeding.

Recently, we successfully knock out several cotton genes by CRISPR-Cas 9 system with an average 65-85% efficiency. We also perform the whole genome sequencing to investigate the off-target in the CRISPR-Cas9 edited cotton plants. The results showed that of 2000+ potential off-targets sites (allowing ≤ 5 mismatches within the 20-bp sgRNA and 3-bp PAM sequences), the WGS data revealed that only a few （less than 10) are bona fide off-target mutations which suggested that CRISPR/Cas9 system is highly specific for the editing of genes of polyploid plant species.Then, we further developed a high-throughput genome editing system in cotton. A sgRNAs library (containing 1100 sgRNAs targeted to 600 independent genes ) was constructed and cloned into the CRISPR-Cas 9 vector. By this way, we can edit several hundred target genes in one transformation. This system need a very high efficient cotton genetic transformation system to generate thousands of regenerated plantlets by somatic embryogenesis. The data we obtained recently suggested that this system works pretty well in cotton. In addition, we start to work a new genome editing system in cotton by using a Francisella novicida (Fn) CRISPR-Cpf1-based genome-editing method. Cpf1, a single-strand RNA-guided endonuclease of the class 2 CRISPR-Cas system that cleaves targeted DNA with features distinct from those of Cas9. For example, preferring a T-rich protospacer-adjencent motif (PAM) and cutting in staggered ends. This system has several advantages over the CRISPR-Cas 9 system including small Cas protein size, generating sticky end after cleaving the DNA, cutting the RNA target and lower off-target risk. Our data showed that CRISPR-Cpf1 system works very well with high efficiency and accuracy. At the same time, we developed a base editing system for cotton by fusion of nuclease-inactive clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (dCas9) with activation-induced cytidine deaminase (AID). Guided by single guide (sg)RNAs, dCas9-AID will directly transfer cytidines or guanines (C or G) to the other three bases independent of AID hotspot motifs, generating a large repertoire of variants at desired loci. Coupled with a uracil-DNA glycosylase inhibitor, dCas9- AIDx converted targeted cytidines (C) especifically to thymines (T), creating specific point mutations. By targeting GhCLA1 with dCas9-AID, we efficiently created known and new mutations showing various phenotype . Thus, targeted AID-mediated mutagenesis (TAM) provides a forward genetic tool to screen for gain-of-function variants at base resolution in cotton.

Developing tolerant cultivars by incorporating resistant genes is regarded as a potential strategy for controlling Verticillium wilt that causes severe losses in the yield and fiber quality of cotton. Here, we identified the gene GbHyPRP1 in Gossypium barbadense, which encodes a protein containing both proline-rich repetitive and Pollen Ole e I domains. GbHyPRP1 is located in the cell wall. The transcription of this gene mainly occurs in cotton roots and stems, and is drastically down-regulated upon infection with Verticillium dahliae. Silencing HyPRP1 dramatically enhanced cotton resistance to V. dahliae. Over-expression of HyPRP1 significantly compromised the resistance of transgenic Arabidopsis plants to V. dahliae. The GbHyPRP1 promoter region contained several putative phytohormone-responsive elements, of which SA was associated with gene down-regulation. We compared the mRNA expression patterns of HyPRP1-silenced plants and the WT at the global level by RNA-Seq. A total of 1735 unique genes exhibited significant differential expression. Of these, 79 DEGs involved in cell wall biogenesis and 43 DEGs associated with the production of ROS were identified. Further, we observed a dramatic thickening of interfascicular fibers and vessel walls and an increase in lignin the HyPRP1-silenced cotton plants compared with the WT after inoculation with V. dahliae. Additionally, silencing of HyPRP1 markedly enhanced ROS accumulation in the root tips of cotton inoculated with V. dahliae. Taken together, our results suggest that HyPRP1 performs a role in the negative regulation of cotton resistance to V. dahliae via the thickening of cell walls and ROS accumulation. *For correspondence: Zhi-ying, Ma; Acknowledgments: This research was financially supported by the National Natural Science Foundation of China (No. 31301370) and Science and Technology Support Program of Hebei Province (16226307D).

Systematic analysis of cotton non-specific lipid transfer proteins revealed a clade of genes that play an important role in fiber development Cheng-sheng Meng1,2, Yuan-yuan Yan1,2, Zheng-wen Liu1, Li-ting Chen1, Yan Zhang1, Xiu-xin Li1, Li-qiang Wu1, Gui-yin Zhang1, Xing-fen Wang1* and Zhi-ying Ma1* 1College of Agronomy, North China Key Laboratory for Germplasm Resources of Education Ministry, Co-Innovation Center For Cotton Industry of Hebei Province, Hebei Agricultural University, Baoding, Hebei 071001, China 2 These authors contributed equally to this work *Correspondence: mzhy@hebau.edu.cn; cotton@hebau.edu.cn It makes great sense to investigate the regulatory mechanism of fiber development of cotton as it supports an important economic industry-textile. Plant non-specific lipid transfer proteins (nsLTPs) are capable to bind in vitro to various phospholipids including phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol and galactolipids with broad specificity. Multiple physiological functions of nsLTPs have been suggested, including membrane and liposome biogenesis, somatic embryogenesis, pollen development, stress resistance, defence and signal transduction. Although some nsLTPs were isolated from cotton fibers and supposed to regulate fiber development, little progress has been achieved on how nsLTPs regulate fiber development. In the present study, nsLTP genes were strictly identified from cotton genome with 138 members in G. hirsutum, 65 members in G. arboreum and 70 members in G. raimondii. These cotton nsLTP genes could be clustered into ten subgroups according to the number of flanking amino acid residues within the conserved ECM domain. Interestingly, type Ⅺ extremely expanded in G. hirsutum genome to be the largest subgroup, which is different from Arabidopsis, cacao，grape， rape and rice whose genome contain most members belonging to type Ⅰ or type Ⅱ. Sequence analysis revealed that GhLtpⅪs were evolutionally close to GhLtpⅡ12-15, and duplication pairs were indentified between GhLtpⅪs and GhLtpⅡs, indicating that type Ⅺ genes of G. hirsutum are likely to diverge from type Ⅱ genes. A large amount of tandem duplication events and non-reciprocal DNA exchanges were found within GhLtpⅪs, which might contribute to the tremendous expansion of type Ⅺ genes. Transcriptional analysis showed that GhLtps were highly transcribed in ovules and fibers, especially GhLtpⅪs whose transcription was significantly higher during fiber elongation in upland cotton cultivars with long fibers, suggesting an essential role of GhLtpⅪs in fiber elongation. Additionally, analysis of the published transcriptome data suggested that most of GhLtpⅪs, ignoring 12 genes that were scarcely detected, were significantly differentially transcribed in ovules at 10 and 20DPA compared with their orthologs in genome A or D, indicating a correlation of cotton type Ⅺ genes with fiber evolution. As fibers are trichomes of the outer epidermis of a single ovule, GhLtpⅪ17, 24, 27 and 28 were cloned and ectopically expressed in Arabidopsis. And significantly elongated trichomes of GhLtpⅪs overexpressed Arabidopsis suggested functional roles of GhLtpⅪs in promoting cell elongation. Our results implied a clade of nsLTP genes regulating fiber elongation and provide new insights into the phenotypic evolution of Gossypium species.