The protein phosphatase (PP2C) gene family, known to participate in cellular processes, is one of the momentous and conserved plant-specific gene families that regulate signal transduction in eukaryotic organisms. Recently, PP2Cs were identified in Arabidopsis and various other crop species, but analysis of PP2C in cotton is yet to be reported. In the current research, we found 87 (Gossypiumarboreum), 147 (Gossypiumbarbadense), 181 (Gossypiumhirsutum), and 99 (Gossypiumraimondii) PP2C-encoding genes in total from the cotton genome. Herein, we provide a comprehensive analysis of the PP2C gene family in cotton, such as gene structure organization, gene duplications, expression profiling, chromosomal mapping, protein motif organization, and phylogenetic relationships of each species. Phylogenetic analysis further categorized PP2C genes into 12 subgroups based on conserved domain composition analysis. Moreover, we observed a strong signature of purifying selection among duplicated pairs (i.e., segmental and dispersed) of Gossypiumhirsutum. We also observed the tissue-specific response of GhPP2C genes in organ and fiber development by comparing the RNA-sequence (RNA-seq) data reported on different organs. The qRT-PCR validation of 30 GhPP2C genes suggested their critical role in cotton by exposure to heat, cold, drought, and salt stress treatments. Hence, our findings provide an overview of the PP2C gene family in cotton based on various bioinformatic tools that demonstrated their critical role in organ and fiber development, and abiotic stress tolerance, thereby contributing to the genetic improvement of cotton for the resistant cultivar.

Verticillium dahliae, a destructive and soil-borne fungal pathogen, causes massive losses in cotton yields. However, the resistance mechanism to V. dahilae in cotton is still poorly understood. Accumulating evidence indicates that chitinases are crucial hydrolytic enzymes, which attack fungal pathogens by catalyzing the fungal cell wall degradation. As a large gene family, to date, the chitinase genes (Chis) have not been systematically analyzed and effectively utilized in cotton. Here, we identified 47, 49, 92 and 116 Chis from four sequenced cotton species, diploid Gossypium raimondii (D₅), G. arboreum (A₂), tetraploid G. hirsutum acc. TM-1 (AD₁) and G. barbadense acc. 3–79 (AD₂), respectively. The orthologous genes were not one-to-one correspondence in the diploid and tetraploid cotton species, implying changes in the number of Chis in different cotton species during the evolution of Gossypium. Phylogenetic classification indicated that these Chis could be classified into six groups, with distinguishable structural characteristics. The expression patterns of Chis indicated their various expressions in different organs and tissues and in the V. dahliae response. Silencing of Chi23, Chi32, or Chi47 in cotton significantly impaired the resistance to V. dahliae, suggesting these genes might act as positive regulators in disease resistance to V. dahliae.

Down-regulation of 5-aminolevulinic acid dehydratase encoding genes results in elevated levels of reactive oxygen species, salicylic acid and resistance to Vert

Lysin motif (LysM)-containing proteins are important pattern recognition receptors (PRRs) in plants, which function in the perception of microbe-associated molecular patterns (MAMPs) and in the defense against pathogenic attack. To date, the LysM genes have not been systematically analyzed in cotton or effectively utilized for disease resistance. Here, we identified 29, 30, 60, and 56 LysM genes in the four sequenced cotton species, diploid Gossypium raimondii, diploid G. arboreum, tetraploid G. hirsutum acc. TM-1, and G. barbadense acc. 3–79, respectively. These LysM genes were classified into four groups with different structural characteristics and a variety of expression patterns in different organs and tissues when induced by chitin or Verticillium dahliae. We further characterized three genes, Lyp1, Lyk7 and LysMe3, which showed significant increase in expression in response to chitin signals, V. dahliae challenge and several stress-related signaling compounds. Lyp1, Lyk7 and LysMe3 proteins were localized to the plasma membrane, and silencing of their expression in cotton drastically impaired salicylic acid, jasmonic acid, and reactive oxygen species generation, impaired defense gene activation, and compromised resistance to V. dahliae. Our results indicate that Lyp1, Lyk7, and LysMe3 are important PRRs that function in the recognition of chitin signals to activate the downstream defense processes and induce cotton defense mechanisms against V. dahliae.

Glucose-6-phosphate dehydrogenase (G6PDH) is known as a critical enzyme responsible for nicotinamide adenine dinucleotide phosphate (NADPH) generation in the pentose phosphate pathway (PPP), and has an essential function in modulating redox homeostasis and stress responsiveness. In the present work, we characterized the nine members of the G6PDH gene family in soybean. Phylogenic analysis and transit peptide prediction showed that these soybean G6PDHs are divided into plastidic (P) and cytosolic (Cy) isoforms. The subcellular locations of five GmG6PDHs were further verified by confocal microscopy in Arabidopsis mesophyll protoplasts. The respective GmG6PDH genes had distinct expression patterns in various soybean tissues and at different times during seed development. Among them, the Cy-G6PDHs were strongly expressed in roots, developing seeds and nodules, while the transcripts of P-G6PDHs were mainly detected in green tissues. In addition, the activities and transcripts of GmG6PDHs were dramatically stimulated by different stress treatments, including salt, osmotic and alkali. Notably, the expression levels of a cytosolic isoform (GmG6PDH2) were extraordinarily high under salt stress and correlated well with the G6PDH enzyme activities, possibly implying a crucial factor for soybean responses to salinity. Enzymatic assay of recombinant GmG6PDH2 proteins expressed in Escherichia coli showed that the enzyme encoded by GmG6PDH2 had functional NADP⁺-dependent G6PDH activity. Further analysis indicated overexpression of GmG6PDH2 gene could significantly enhance the resistance of transgenic soybean to salt stress by coordinating with the redox states of ascorbic acid and glutathione pool to suppress reactive oxygen species generation. Together, these results indicate that GmG6PDH2 might be the major isoform for NADPH production in PPP, which is involved in the modulation of cellular AsA-GSH cycle to prevent the oxidative damage induced by high salinity.

A fungal effector that is toxic to plant cells was identified in Verticillium dahliae. The effector contains a non-canonical Common in several Fungal Extracellular Membrane proteins (CFEM) domain, a tandem repeat region consisting of four 14-amino acid repeats rich in proline, and a C-terminal region, thus is designated V. dahliae tetrapeptide repeat protein (VdTRP). The membrane targeting of VdTRP is vital for its cell toxicity. CFEM mediates the membrane targeting and the tandem repeat region exerts the toxic function upon cell membrane. The chitinase-like 1 (CTL1), an essential apoplastic protein of cotton, can redirect VdTRP from cell membrane to apoplast. Transgenic cotton overexpressing CTL1 greatly enhances cotton resistance to V. dahliae without affecting cotton growth and development, implicating its potential application in breeding cotton with high wilt resistance. Our data demonstrates that genetic manipulation of effector target constitutes potential strategy for improving crop resistance to fungal pathogens.