Cotton GhNAC4 promotes drought tolerance by regulating secondary cell wall biosynthesis and ribosomal protein homeostasis.

Drought has a severe impact on the quality and yield of cotton. Deciphering the key genes related to drought tolerance is important for understanding the regulation mechanism of drought stress and breeding drought-tolerant cotton cultivars. Several studies have demonstrated that NAC transcription factors are crucial in the regulation of drought stress, however, the related functional mechanisms are still largely unexplored. Here, we identified that NAC transcription factor GhNAC4 positively regulated drought stress tolerance in cotton. The expression of GhNAC4 was significantly induced by abiotic stress and plant hormones. Silencing of GhNAC4 distinctly impaired the resistance to drought stress and overexpressing GhNAC4 in cotton significantly enhanced the stress tolerance. RNA-seq analysis revealed that overexpression of GhNAC4 enriched the expression of genes associated with the biosynthesis of secondary cell walls and ribosomal proteins. We confirmed that GhNAC4 positively activated the expressions of GhNST1, a master regulator reported previously in secondary cell wall formation, and two ribosomal protein-encoding genes GhRPL12 and GhRPL18p, by directly binding to their promoter regions. Overexpression of GhNAC4 promoted the expression of downstream genes associated with the secondary wall biosynthesis, resulting in enhancing secondary wall deposition in the roots, and silencing of GhRPL12 and GhRPL18p significantly impaired the resistance to drought stress. Taken together, our study reveals a novel pathway mediated by GhNAC4 that promotes secondary cell wall biosynthesis to strengthen secondary wall development and regulates the expression of ribosomal protein-encoding genes to maintain translation stability, which ultimately enhances drought tolerance in cotton.

Revealing the Complete Bispecific Phosphatase Genes (DUSPs) across the Genome and Investigating the Expression Patterns of GH_A11G3500 Resistance against Verticillium wilt.

DUSPs, a diverse group of protein phosphatases, play a pivotal role in orchestrating cellular growth and development through intricate signaling pathways. Notably, they actively participate in the MAPK pathway, which governs crucial aspects of plant physiology, including growth regulation, disease resistance, pest resistance, and stress response. DUSP is a key enzyme, and it is the enzyme that limits the rate of cell metabolism. At present, complete understanding of the DUSP gene family in cotton and its specific roles in resistance to Verticillium wilt (VW) remains elusive. To address this knowledge gap, we conducted a comprehensive identification and analysis of four key cotton species: Gossypium arboreum, Gossypium barbadense, Gossypium hirsutum, and Gossypium raimondii. The results revealed the identification of a total of 120 DUSP genes in the four cotton varieties, which were categorized into six subgroups and randomly distributed at both ends of 26 chromosomes, predominantly localized within the nucleus. Our analysis demonstrated that closely related DUSP genes exhibited similarities in terms of the conserved motif composition and gene structure. A promoter analysis performed on the GhDUSP gene promoter revealed the presence of several cis-acting elements, which are associated with abiotic and biotic stress responses, as well as hormone signaling. A tissue expression pattern analysis demonstrated significant variations in GhDUSP gene expression under different stress conditions, with roots exhibiting the highest levels, followed by stems and leaves. In terms of tissue-specific detection, petals, leaves, stems, stamens, and receptacles exhibited higher expression levels of the GhDUSP gene. The gene expression analysis results for GhDUSPs under stress suggest that DUSP genes may have a crucial role in the cotton response to stress in cotton. Through Virus-Induced Gene Silencing (VIGS) experiments, the silencing of the target gene significantly reduced the resistance efficiency of disease-resistant varieties against Verticillium wilt (VW). Consequently, we conclude that GH_A11G3500-mediated bispecific phosphorylated genes may serve as key regulators in the resistance of G. hirsutum to Verticillium wilt (VW). This study presents a comprehensive structure designed to provide an in-depth understanding of the potential biological functions of cotton, providing a strong foundation for further research into molecular breeding and resistance to plant pathogens.

Complete genome sequence of a novel virus belonging to the genus Badnavirus in jujube (Ziziphus jujuba Mill.) in China.

The genome sequence of a novel circular DNA virus related to members of the genus Badnavirus was identified in diseased jujube trees by high-throughput sequencing and verified by conventional Sanger sequencing of cloned PCR products. The name "jujube badnavirus WS" (JuBWS) is proposed for this virus. Diseased jujube leaves showed yellow mosaic and malformation symptoms, with round chlorotic spots found on diseased fruit. The genome of this virus has a length of 6450 nt and has a typical badnavirus genomic structure with three open reading frames (ORFs). JuBWS was identified as a novel badnavirus based on nucleotide differences in the RNase (RT + RNase H) coding region of ORF3. The JuBWS sequence showed 70.48-76.41% nucleotide sequence identity to other known badnaviruses, thus meeting the taxonomic criterion for establishing a new species within the genus Badnavirus. This study suggested that the novel badnavirus might be a pathogen associated with jujube mosaic disease, and this will be investigated in the future.

Genetic Diversity and Pathogenicity of Verticillium dahliae Isolates and Their Co-occurrence with Fusarium oxysporum f. sp. vasinfectum Causing Cotton Wilt in Xinjiang, China.

Cotton production in Xinjiang, the largest cotton-producing area in China, has an increasingly serious disease threat from Verticillium dahliae. Eighty-five V. dahliae isolates were obtained from wilted cotton plants collected from eight counties in Xinjiang. The isolates were assessed for genotypic diversity by DNA sequence analysis and PCR molecular genotyping with specific markers for race 1, race 2, defoliating (D) pathotype, nondefoliating (ND) pathotype, and mating type idiomorph Mat1-2. Isolates belonged to lineages 1A or 2B, with three subgenotypes found in each lineage. All isolates tested positive for race 2 and Mat1-2 markers. All isolates in lineage 2B tested positive for the ND pathotype marker but only isolates in the major subgenotype in lineage 1A tested positive for the D pathotype marker. Pathogenicity assays on Gossypium hirsutum 'Acala 44' demonstrated no significant difference among subgenotypes within each lineage. Isolates in lineage 1A caused greater shoot weight reductions, percent leaf drop, and percent diseased leaves than isolates in lineage 2B. One isolate in each lineage for 1A and 2B was avirulent. Isolates in lineage 1A caused greater than 50% leaf drop and a 17-g shoot weight reduction compared with a 9% leaf drop and a 6-g shoot weight reduction by isolates in lineage 2B. Overall, 42% of the V. dahliae isolates from Xinjiang were D pathotype but the percentage varied widely among locations. Two plants had both pathotypes. Nineteen isolates of Fusarium oxysporum f. sp. vasinfectum VCG0114 (race 4) also were recovered from wilted plants in Xinjiang. Two plants had both Verticillium wilt and Fusarium wilt pathogens. Both pathogens should be considered when using or developing wilt resistant or tolerant materials for Xinjiang.

Detection and Characterization of Fusarium oxysporum f. sp. vasinfectum VCG0114 (Race 4) Isolates of Diverse Geographic Origins.

A highly virulent cotton wilt pathogen, Fusarium oxysporum f. sp. vasinfectum VCG0114 (race 4) was found in West Texas in 2017, after being known in California since 2001. Isolates obtained from wilted plants collected in 2017 from Texas, in 2015 from China, and during 2001 to 2014 from California and isolates from historical collections including the race 4 reference isolate were characterized by soil-infestation pathogenicity assays, DNA sequence analysis, and vegetative compatibility analysis. All obtained F. oxysporum f. sp. vasinfectum isolates belonged to VCG0114. All of these isolates, except one isolate from China, caused disease in a soil-infestation assay without nematodes. Thus, they belong to the nematode-independent pathotype. Texas isolates were significantly more virulent than were isolates from China or California on Gossypium barbadense 'Pima S-7'. Four different genotypes (N, T, MT, and MiT) were identified based on the transposable element Tfo1 insertion into the PHO gene and independent MULE or MITE insertions into the Tfo1 transposon. Some significant differences in virulence were detected among the genotypes in some locations. No differences in pathogenicity were observed between the California and China collection isolates on Pima S-7, and the virulence of the major genotypes was similar on the Gossypium hirsutum cultivar 'Stoneville 474' or the Barbren 713 germplasm line. Simple polymerase chain reaction (PCR) methods were developed to specifically determine and detect the four genotypes within VCG0114. A specific PCR method to detect all VCG0114 isolates was also developed. These methods will facilitate the timely identification of infested fields and seed lots and the elucidation of evolutionary relationships among the isolates. This should help to closely monitor the movement of the pathogen and reduce dissemination of these devastating pathogens.

Transcriptional responses to drought stress in root and leaf of chickpea seedling.

Chickpea (Cicer arietinum L.) is an important pulse crop grown mainly in the arid and semi-arid regions of the world. Due to its taxonomic proximity with the model legume Medicago truncatula and its ability to grow in arid soil, chickpea has its unique advantage to understand how plant responds to drought stress. In this study, an oligonucleotide microarray was used for analyzing the transcriptomic profiles of unigenes in leaf and root of chickpea seedling under drought stress, respectively. Microarray data showed that 4,815 differentially expressed unigenes were either ≥ 2-fold up- or ≤ 0.5-fold down-regulated in at least one of the five time points during drought stress. 2,623 and 3,969 unigenes were time-dependent differentially expressed in root and leaf, respectively. 110 pathways in two tissues were found to respond to drought stress. Compared to control, 88 and 52 unigenes were expressed only in drought-stressed root and leaf, respectively, while nine unigenes were expressed in both the tissues. 1,922 function-unknown unigenes were found to be remarkably regulated by drought stress. The expression profiles of these time-dependent differentially expressed unigenes were useful in furthering our knowledge of molecular mechanism of plant in response to drought stress.

Quantitative Trait Locus Mapping and Identification of Candidate Genes for Resistance to Fusarium Wilt Race 7 Using a Resequencing-Based High Density Genetic Bin Map in a Recombinant Inbred Line Population of Gossypium barbadense.

Fusarium wilt caused by Fusarium oxysporum f. sp. vasinfectum (FOV) is one of the most destructive diseases in cotton (Gossypium spp.) production, and use of resistant cultivars is the most cost-effective method managing the disease. To understand the genetic basis of cotton resistance to FOV race 7 (FOV7), this study evaluated a recombinant inbred line (RIL) population of 110 lines of G. barbadense from a cross between susceptible Xinhai 14 and resistant 06-146 in eight tests and constructed a high-density genetic linkage map with resequencing-based 933,845 single-nucleotide polymorphism (SNP) markers covering a total genetic distance of 2483.17 cM. Nine quantitative trait loci (QTLs) for FOV7 resistance were identified, including qFOV7-D03-1 on chromosome D03 in two tests. Through a comparative analysis of gene expression and DNA sequence for predicted genes within the QTL region between the two parents and selected lines inoculated with FOV7, GB_D03G0217 encoding for a calmodulin (CaM)-like (CML) protein was identified as a candidate gene. A further analysis confirmed that the expression of GB_D03G0217 was suppressed, leading to increased disease severity in plants of the resistant parent with virus induced gene silencing (VIGS).

The susceptibility of sea-island cotton recombinant inbred lines to Fusarium oxysporum f. sp. vasinfectum infection is characterized by altered expression of long noncoding RNAs.

Disease resistance is one of the most complicated yet important plant traits. The potential functions of long noncoding RNAs (lncRNAs) in response to pathogenic fungi remain unclear. In this study, we sequenced the transcriptomes of four different sea-island cotton (Gossypium barbadense) recombinant inbred lines (RILs) with susceptible, highly susceptible, highly resistant, or super highly resistant phenotypes and compared their responses to Fusarium oxysporum f. sp. vasinfectum (Fov) infection with those of their susceptible and resistant parents. Infection-induced protein coding genes were highly enriched in similar disease resistance-related pathways regardless of fungal susceptibility. In contrast, we found that the expression of a large number of Fov infection-induced lncRNAs was positively correlated with plant susceptibility. Bioinformatics analysis of potential target mRNAs of lncRNAs with both trans-acting and cis-acting mechanisms showed that mRNAs co-expressed or co-located with Fov-regulated lncRNAs were highly enriched in disease resistance-related pathways, including glutathione metabolism, glycolysis, plant hormone signal transduction, anthocyanin biosynthesis, and butanoate metabolism. Together these results suggest that lncRNAs could play a significant role in the response to pathogenic fungal infection and the establishment of disease resistance. The transcriptional regulation of these infection-susceptible lncRNAs could be coordinated with infection-susceptible mRNAs and integrated into a regulatory network to modulate plant-pathogen interactions and disease resistance. Fov-susceptible lncRNAs represent a novel class of molecular markers for breeding of Fov-resistant cotton cultivars.

A cupin domain is involved in α-amylase inhibitory activity.

Proteinaceous α-amylase inhibitors have specialized activities that make some strong inhibition of α-amylases. New α-amylase inhibitors continue to be discovered so far. A proteinaceous α-amylase inhibitor CL-AI was isolated and identified from chickpea seeds. CL-AI, encoded by Q9SMJ4, was a storage legumin precursor containing one α-chain and one ß-chain, and each chain possessed a same conserved cupin domain. Amino acid mutation and deficiency of cupin domain would lead to loss of α-amylase inhibitory activity, indicating that it was essential for inhibitory activity. CL-AI(α + ß) in its single stranded state in vivo had inhibitory activity. After it was processed into one α-chain and one ß-chain, the two chains were connected to each other via disulfide bond, which would cover the cupin domains and lead to the loss of inhibitory activity. The CL-AI(α + ß), α-chain and ß-chain could inhibit various α-amylases and delay the seed germination of wheat, rice and maize as well as the growth and development of potato beetle larva. Two cupin proteins, Glycinin G1 in soybean and Glutelinin in rice were also found to have inhibitory activity. Our results indicated that the cupin domain is involved in α-amylase inhibitory activity and the proteins with a cupin domain may be a new kind of proteinaceous α-amylase inhibitor.

Isolation, Identification and Characterization of a New Type of Lectin with α-Amylase Inhibitory Activity in Chickpea (Cicer arietinum L.).

BACKGROUND: Plant lectins are a group of highly diverse proteins that possess at least one non-catalytic domain that binds reversibly to a specific mono- or oligosaccharide. So far, only seven members in the lectin-arcelin-αAI1 supergene family in legume lectins have been reported to have inhibitory activity of α-amylases. OBJECTIVE & METHODS: A proteinaceous α-amylase inhibitor was isolated and purified using Ammonium sulfate precipitation (ASP), Ion exchange chromatography (IEC) and Reversed phase liquid chromatography (RPLC) from the mature seeds of chickpea. RESULTS & CONCLUSION: Identification by Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS-MS) indicated that the purified proteinaceous α-amylase inhibitor was a chickpea lectin CAL in GenBank (accession No. AGL46982.1). CAL had 227 aa containing a hemopexin- like repeats domain and was a cytoplasm protein. It had very low (<17%) identity with seven members in the lectin-arcelin-aAI1 supergene family in legumes that have α-amylase inhibitory activity. The purified CAL derived from prokaryotic expression was confirmed to have inhibitory activity against various α-amylases. The inhibitory activity of CAL against various α-amylases was severely affected by temperature, pH, incubation time, substrate concentration and CAL protein concentration. Feeding CAL reduced the weight of potato beetle larvae by 27.21% (P<0.05) and survival rate by 6.67% (P>0.05). Our results indicated that CAL is a new type of lectin with inhibitory activity against α-amylases in legume lectins, which can be used as a candidate in genetic engineering for breeding for pest resistance.

Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene.

A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1I-type gene fragments. Amplification products were digested with the Bsp119I and BanI enzymes, and four kinds of known cry1I-type genes were successfully identified. The results showed that cry1I-type genes appeared in 95 of 115 B. thuringiensis isolates and 7 of 13 standard strains. A novel cry1I-type gene was found in one standard strain and six isolates. The novel cry1I gene was cloned from B. thuringiensis isolate Btc007 and subcloned into vector pET-21b. Then it was overexpressed in Escherichia coli BL21(DE3). The expressed product was shown to be toxic to the diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia furnacalis), and soybean pod borer (Leguminivora glycinivorella). However, it was not toxic to the cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), or elm leaf beetle (Pyrrhalta aenescens) in bioassays. Subsequently, the Cry protein encoded by this novel cry gene was designated Cry1Ie1 by the B. thuringiensis delta-endotoxin nomenclature committee.