Acetylation of GhCaM7 enhances cotton resistance to Verticillium dahliae.

Protein lysine acetylation is an important post-translational modification mechanism involved in cellular regulation in eukaryotes. Calmodulin (CaM) is a ubiquitous Ca2+ sensor in eukaryotes and is crucial for plant immunity, but it is so far unclear whether acetylation is involved in CaM-mediated plant immunity. Here, we found that GhCaM7 is acetylated upon Verticillium dahliae (V. dahliae) infection and a positive regulator of V. dahliae resistance. Overexpressing GhCaM7 in cotton and Arabidopsis enhances V. dahliae resistance and knocking-down GhCaM7 makes cotton more susceptible to V. dahliae. Transgenic Arabidopsis plants overexpressing GhCaM7 with mutation at the acetylation site are more susceptible to V. dahliae than transgenics overexpressing the wild-type GhCaM7, implying the importance of the acetylated GhCaM7 in response to V. dahliae infection. Yeast two-hybrid, bimolecular fluorescent complementation, luciferase complementation imaging, and coimmunoprecipitation assays demonstrated interaction between GhCaM7 and an osmotin protein GhOSM34 that was shown to have a positive role in V. dahliae resistance. GhCaM7 and GhOSM34 are co-localized in the cell membrane. Upon V. dahliae infection, the Ca2+ content reduces almost instantly in plants with downregulated GhCaM7 or GhOSM34. Down regulating GhOSM34 enhances accumulation of Na+ and increases cell osmotic pressure. Comparative transcriptomic analyses between cotton plants with an increased or reduced expression level of GhCaM7 and wild-type plants indicate the involvement of jasmonic acid signaling pathways and reactive oxygen species in GhCaM7-enabled disease resistance. Together, these results demonstrate the involvement of CaM protein in the interaction between cotton and V. dahliae, and more importantly, the involvement of the acetylated CaM in the interaction.

Regulation of carbohydrate metabolism during anther development in a thermo-sensitive genic male-sterile wheat line.

Bainong sterility (BNS) is a thermo-sensitive genic male sterile wheat line, characterised by anther fertility transformation in response to low temperature (LT) stress during meiosis, the failure of vacuole decomposition and the absence of starch accumulation in sterile bicellular pollen. Our study demonstrates that the late microspore (LM) stage marks the transition from the anther growth to anther maturation phase, characterised by the changes in anther structure, carbohydrate metabolism and the main transport pathway of sucrose (Suc). Fructan is a main storage polysaccharide in wheat anther, and its synthesis and remobilisation are crucial for anther development. Moreover, the process of pollen amylogenesis and the fate of the large vacuole in pollen are closely intertwined with fructan synthesis and remobilisation. LT disrupts the normal physiological metabolism of BNS anthers during meiosis, particularly affecting carbohydrate metabolism, thus determining the fate of male gametophytes and pollen abortion. Disruption of fructan synthesis and remobilisation regulation serves as a decisive event that results in anther abortion. Sterile pollen exhibits common traits of pollen starvation and impaired starch accumulation due to the inhibition of apoplastic transport starting from the LM stage, which is regulated by cell wall invertase TaIVR1 and Suc transporter TaSUT1.

A wheat WRKY transcription factor TaWRKY17 enhances tolerance to salt stress in transgenic Arabidopsis and wheat plant.

WRKY transcription factors are essential to plant growth, development, resistance, and the regulation of metabolic pathways. In this study, we characterized TaWRKY17, a WRKY transcription factor from wheat, which was differentially expressed in various wheat organs and was up-regulated by salt, drought, hydrogen peroxide (H2O2) and abscisic acid (ABA) treatment. To analyze TaWRKY17 function under salt stress, we obtained stable T3 generation transgenic Arabidopsis and wheat TaWRKY17 overexpression plants. TaWRKY17 overexpression in Arabidopsis and wheat caused a significant plant salt-stress tolerance enhancement. Under salt stress, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) enzyme activities were elevated in transgenic Arabidopsis and wheat plants compared with the wild type (WT), whereas H2O2 and malondialdehyde (MDA) accumulation was reduced in the transgenic lines. Moreover, ABA/reactive oxygen species (ROS)-related, and stress-response genes were regulated in the transgenic wheat plants, increasing tolerance to salt stress. The transgenic wheat plants were highly sensitive to ABA during seed germination and early seedling growth. In addition, TaWRKY17 virus-induced gene silencing (VIGS) decreased salt tolerance. These results showed that TaWRKY17 enhances salt tolerance by regulating ABA/ROS-related, and stress-response genes and increasing anti-oxidative stress capabilities. Therefore, this gene could be a target for the genetic modification of wheat.

A rapid, simple, and highly efficient method for VIGS and in vitro-inoculation of plant virus by INABS applied to crops that develop axillary buds and can survive from cuttings.

BACKGROUND: Virus-induced gene silencing (VIGS) is one of the most convenient and powerful methods of reverse genetics. In vitro-inoculation of plant virus is an important method for studying the interactions between viruses and plants. Agrobacterium-based infiltration has been widely adopted as a tool for VIGS and in vitro-inoculation of plant virus. Most agrobacterium-based infiltration methods applied to VIGS and virus inoculation have the characteristics of low transformation efficiencies, long plant growth time, large amounts of plant tissue, large test spaces, and complex preparation procedures. Therefore, a rapid, simple, economical, and highly efficient VIGS and virus inoculation method is in need. Previous studies have shown that the selection of suitable plant tissues and inoculation sites is the key to successful infection. RESULTS: In this study, Tobacco rattle virus (TRV) mediated VIGS and Tomato yellow leaf curl virus (TYLCV) for virus inoculation were developed in tomato plants based on the agrobacterium tumefaciens-based infiltration by injection of the no-apical-bud stem section (INABS). The no-apical-bud stem section had a "Y- type" asymmetric structure and contained an axillary bud that was about 1-3 cm in length. This protocol provides high transformation (56.7%) and inoculation efficiency (68.3%), which generates VIGS transformants or diseased plants in a very short period (8 dpi). Moreover, it greatly reduces the required experimental space. This method will facilitate functional genomic studies and large-scale disease resistance screening. CONCLUSIONS: Overall, a rapid, simple, and highly efficient method for VIGS and virus inoculation by INABS was developed in tomato. It was reasonable to believe that it can be used as a reference for the other virus inoculation methods and for the application of VIGS to other crops (such as sweet potato, potato, cassava and tobacco) that develop axillary buds and can survive from cuttings.

A Ricin B-Like Lectin Protein Physically Interacts with TaPFT and Is Involved in Resistance to Fusarium Head Blight in Wheat.

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, has become one of the most serious diseases that damage wheat. The TaPFT (pore-forming toxin-like) and TaHRC (histidine-rich calcium-binding protein) genes at the quantitative trait locus Fhb1 were identified to confer resistance to FHB in the wheat cultivar Sumai 3. In this study, a wheat ricin B-like lectin gene (designated TaRBL) that interacted with TaPFT was isolated by a yeast two-hybrid screen of a wheat cDNA library. A yeast two-hybrid and bimolecular fluorescence complementation study further verified that TaRBL interacted with TaPFT but not with TaHRC. Gene expression studies showed that upon F. graminearum infection, TaRBL expression was upregulated in resistant cultivars but downregulated in susceptible cultivars. Furthermore, knockdown of TaRBL expression by barley stripe mosaic virus-induced gene silencing significantly reduced the resistance of wheat to FHB in both the resistant cultivar Sumai 3 and the susceptible cultivar Jimai 22. Thus, we conclude that TaRBL encodes a ricin B-like lectin protein that interacts with TaPFT and is involved in resistance to FHB in wheat.

Biological Characteristics and Molecular Mechanism of Fludioxonil Resistance in Botrytis cinerea From Henan Province of China.

The gray mold caused by Botrytis cinerea has a significant impact on tomato production throughout the world. Although the synthetic fungicide fludioxonil can effectively control B. cinerea, there have been several reports of resistance to this fungicide. This study indicated that all of the fludioxonil-resistant strains tested, including one field-resistant isolate and four laboratory strains, had reduced fitness relative to sensitive isolates. In addition to having reduced growth, sporulation, and pathogenicity, the resistant strains were more sensitive to osmotic stress and had significantly (P < 0.05) higher peroxidase activity. BOs1, a kinase in the high-osmolarity glycerol stress response signal transduction pathway, is believed to harbor mutations related to fludioxonil resistance. Sequence analysis of their BOs1 sequences indicated that the fludioxonil-resistant field isolate, XXtom1806, had four point mutations resulting in four amino acid changes (I365S, S531G, T565N, and T1267A) and three amino acids (I365S, S531G, and T565N) in the histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis receptors, and phosphatases domain, which associated with fludioxonil binding. Similarly, two of the laboratory strains, XXtom-Lab1 and XXtom-Lab4, had three (Q846S, I1126S, and G415D) and two (P1051S and V1241M) point mutations, respectively. A third strain, XXtom-lab3, had a 52-bp insertion that included a stop codon at amino acid 256. Interestingly, the BOs1 sequence of the fourth laboratory strain, XXtom-lab5, was identical to those of the sensitive isolates, indicating that an alternative resistance mechanism exists. The study also found evidence of positive cross-resistance between fludioxonil and the dicarboximide fungicides procymidone and iprodione, but no cross-resistance was detected with any other fungicides tested, including boscalid, carbendazim, tebuconazole, and fluazinam.

Cyclin-Dependent Kinase Inhibitor Gene TaICK1 acts as a Potential Contributor to Wheat Male Sterility induced by a Chemical Hybridizing Agent.

Heterosis has been widely accepted as an effective strategy to increase yields in plant breeding. Notably, the chemical hybridization agent SQ-1 induces male sterility in wheat, representing a critical potential tool in hybrid seed production. However, the mechanisms underlying the male sterility induced by SQ-1 still remain poorly understood. In this study, a cyclin-dependent kinase inhibitor gene, TaICK1, which encodes a 229 amino acid protein, was identified as a potential contributor to male sterility in common wheat. The expression of TaICK1 was upregulated during the development of anthers in Xinong1376 wheat treated with SQ-1. Meanwhile, the seed setting rate was found to be significantly decreased in TaICK1 transgenic rice. Furthermore, we identified two cyclin proteins, TaCYCD2;1 and TaCYCD6;1, as interactors through yeast two-hybrid screening using TaICK1 as the bait, which were validated using bimolecular fluorescence complementation. Subcellular localization revealed that the proteins encoded by TaICK1, TaCYCD2;1, and TaCYCD6;1 were localized in the cell nucleus. The expression levels of TaCYCD2;1 and TaCYCD6;1 were lower in Xinong1376 treated with SQ-1. A further analysis demonstrated that the expression levels of OsCYCD2;1 and OsCYCD6;1 were lower in transgenic TaICK1 rice lines as well. Taken together, these results suggest that the upregulation of TaICK1, induced by SQ-1, may subsequently suppress the expression of TaCYCD2;1 and TaCYCD6;1 in anthers, resulting in male sterility. This study provides new insights into the understanding of SQ-1-induced wheat male sterility, as well as the developmental mechanisms of anthers.

Hyperbolic graph convolutional neural network with contrastive learning for automated ICD coding.

The International Classification of Diseases (ICD) is a widely used criterion for disease classification, health monitoring, and medical data analysis. Deep learning-based automated ICD coding has gained attention due to the time-consuming and costly nature of manual coding. The main challenges of automated ICD coding include imbalanced label distribution, code hierarchy and noisy texts. Recent works have considered using code hierarchy or description for better label representation to solve the problem of imbalanced label distribution. However, these methods are still ineffective and redundant since they only interact with a constant label representation. In this work, we introduce a novel Hyperbolic Graph Convolutional Network with Contrastive Learning (HGCN-CL) to solve the above problems and the shortcomings of the previous methods. We adopt a Hyperbolic graph convolutional network on ICD coding to capture the hierarchical structure of codes, which can solve the problem of large distortions when embedding hierarchical structure with graph convolutional network. Besides, we introduce contrastive learning for automatic ICD coding by injecting code features into text encoder to generate hierarchical-aware positive samples to solve the problem of interacting with constant code features. We conduct experiments on the public MIMIC-III and MIMIC-II datasets. The results on MIMIC III show that HGCN-CL outperforms previous state-of-art methods for automatic ICD coding, which achieves a 2.7% and 3.6% improvement respectively compared to previous best results (Hypercore). We also provide ablation experiments and hierarchy visualization to verify the effectiveness of components in our model.

Wheat WRKY transcription factor TaWRKY24 confers drought and salt tolerance in transgenic plants.

Drought and salt stress are major environmental conditions that severely limit plant growth and productivity. WRKY transcription factors play a vital role in the responses against biotic or abiotic stress. In this study, TaWRKY24, a gene of the IIe WRKY family identified in wheat, was cloned and characterized. TaWRKY24 was mainly expressed in wheat leaf and stem and induced by treatment with PEG6000, salt, H2O2, ABA, MeJA, and ethrel. TaWRKY24 transient expression in onion epidermal cells suggested its nuclear localization and its transcriptional activation capability characteristics. Overexpression of TaWRKY24 in tobacco improved the seed germination rate and root growth of seedlings in transgenic lines when subjected to higher mannitol and NaCl concentrations. Further research showed that transgenic lines had higher proline and soluble sugars and lower levels of reactive oxygen species (ROS) and malondialdehyde (MDA). Moreover, compared to normal and negative control plants, TaWRKY24 silenced wheat seedlings had reduced growth under salt and drought stress. This study shows that wheat TaWRKY24 is crucial to plant stress, providing an excellent candidate gene for wheat resistance breeding.

Genome-wide identification of ETHYLENE INSENSITIVE 2 in Triticeae species reveals that TaEIN2-4D.1 regulates cadmium tolerance in Triticum aestivum.

ETHYLENE INSENSITIVE 2 (EIN2), as the core component of the ethylene signaling pathway, can widely regulate plant growth, development, and stress responses. However, the comprehensive study and function of EIN2 in wheat Cadmium (Cd) stress remain largely unexplored. Here, we identified 33 EIN2 genes and designated as TaEIN2-2B to TaEIN2-Un.3 in Triticum aestivum. The analysis of cis-regulatory elements in promoter regions and RNA-Seq showed that TaEIN2s were functionally related to plant growth and development, as well as the response to biotic and abiotic stress. qRT-PCR analysis of TaEIN2s indicated their sensitivity to Cd stress. Compared with WT plants, TaEIN2-4D.1-RNAi transgenic wheat lines showed enhanced shoot and root elongation, dry weight and chlorophyll accumulation, together with a reduced accumulation of Cd in wheat grain. In addition, TaEIN2-4D.1-RNAi transgenic wheat lines showed enhanced Reactive Oxygen Species (ROS) scavenging capacity compared with WT plants. In conclusion, our research indicates that TaEIN2 plays a key role in response to cadmium stress in wheat, which provides valuable information for crop improvement.

Genome-wide identification of xylan glucuronosyltransferase family in cotton and function characterization of GhGUX5 in regulating Verticillium wilt resistance.

Xylan glucuronosyltransferase (GUX) is widely involved in a variety of physiological processes in plants, including plant development, growth and the defense response to pathogens. However, the function of GUX regulators in Verticillium dahliae (V. dahliae) infection has not been considered previously in cotton. Overall, 119 GUX genes were identified from multiple species and were phylogenetically categorized into seven classes. Duplication event analysis indicated that GUXs in Gossypium hirsutum primarily originated from segmental duplication. GhGUXs promoter analysis indicated cis-regulatory elements capable of reacting to several different stresses. RNA-Seq data and qRT-PCR analysis both indicated that most GhGUXs were associated with V. dahliae infection. Gene interaction network analysis showed that GhGUX5 interacted with 11 proteins, and the relative expression of these 11 proteins changed significantly following V. dahliae infection. In addition, silencing and overexpression of GhGUX5 results to enhance and reduce plant's susceptibility to V. dahliae. Further study showed that TRV:GhGUX5 silenced cotton plants exhibited a decrease in the degree of lignification, total lignin content, gene expression levels involved in lignin biosynthesis, and enzyme activity compared with TRV:00. The above results indicate that GhGUX5 enhances Verticillium wilt resistance through the lignin biosynthesis pathway.

Genome-Wide Analysis of Serine Hydroxymethyltransferase Genes in Triticeae Species Reveals That TaSHMT3A-1 Regulates Fusarium Head Blight Resistance in Wheat.

Serine hydroxymethyltransferase (SHMT) plays a pivotal role in cellular one-carbon, photorespiration pathways and it influences the resistance to biotic and abiotic stresses. However, the function of SHMT proteins in wheat remains largely unexplored. In the present study, SHMT genes in five Triticeae species, Oryza sativa, and four dicotyledon species were identified based on whole genome information. The origin history of the target gene was traced by micro-collinearity analysis. Gene expression patterns of TaSHMTs in different tissues, various biotic stresses, exogenous hormones, and two biotic stresses were determined by Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The function of the selected TaSHMT3A-1 was studied by barley stripe mosaic virus-induced gene silencing in common wheat Bainong207. A total of 64 SHMT members were identified and further classified into two main classes based on the structure of SHMT proteins. The gene structure and motif composition analyses revealed that SHMTs kept relatively conserved within the same subclasses. Interestingly, there was a gene, TdSHMT7B-1, on chromosome 7B of Triticum dicoccoides, but there was no SHMT gene on chromosome 7 of other analyzed Triticeae species; TdSHMT7B-1 had fewer exons and conserved motifs than the genes in the same subclass, suggesting that the gene of TdSHMT7B-1 has a notable evolutionary progress. The micro-collinearity relationship showed that no homologs of TaSHMT3A-1 and its two neighboring genes were found in the collinearity region of Triticum urartu, and there were 27 genes inserted into the collinearity region of T. urartu. Furthermore, qRT-PCR results showed that TaSHMT3A-1 was responsive to abiotic stresses (NaCl and cold), abscisic acid, methyl jasmonate, and hydrogen peroxide. Significantly, upon Fusarium graminearum infection, the expression of TaSHMT3A-1 was highly upregulated in resistant cultivar Sumai3. More importantly, silencing of TaSHMT3A-1 compromises Fusarium head blight resistance in common wheat Bainong207. Our new findings suggest that the TaSHMT3A-1 gene in wheat plays an important role in resistance to Fusarium head blight. This provides a valuable reference for further study on the function of this gene family.

New SFT2-like Vesicle Transport Protein (SFT2L) Enhances Cadmium Tolerance and Reduces Cadmium Accumulation in Common Wheat Grains.

Cadmium (Cd) is one of the most toxic heavy metal elements to the environment, which seriously threatens the safe production of food crops. In this study, we identified a novel function of the cytomembrane TaSFT2L protein in wheat (Triticum aestivum). Expression of the TaSFT2L gene in yeast showed no transport activities for Cd, which could explain the role of TaSFT2L in metal tolerance. It was observed that increased autophagic activity in roots caused by silencing of TaSFT2L enhanced Cd tolerance. Transgenic wheat revealed that RNA interference (RNAi) lines enhanced the wheat growth concerning the increased shoot or root elongation, dry weight, and chlorophyll accumulation. Furthermore, RNAi lines decreased root-to-grain Cd translocation in wheat by nearly 68% and Cd accumulation in wheat grains by 53%. Meanwhile, the overexpression lines displayed a compromised growth response and increased Cd accumulation in wheat tissues, compared to wild type. These findings show that TaSFT2L is a key gene involved in regulation of Cd translocation in wheat, and its silencing to form transgenic wheat can inhibit Cd accumulation. This has the ability to alleviate the food chain-associated impact of environmental pollution on human health.

Clay nanosheet-mediated delivery of recombinant plasmids expressing artificial miRNAs via leaf spray to prevent infection by plant DNA viruses.

Whitefly-transmitted begomoviruses are economically important plant pathogens that cause severe problems in many crop plants, such as tomato, papaya, cotton, and tobacco. Tomato yellow leaf curl virus (TYLCV) is a typical monopartite begomovirus that has been extensively studied, but methods that can efficiently control begomoviruses are still scarce. In this study, we combined artificial microRNA (amiRNA)-mediated silencing technology and clay nanosheet-mediated delivery by spraying and developed a method for efficiently preventing TYLCV infection in tomato plants. We designed three amiRNAs that target different regions of TYLCV to silence virus-produced transcripts. Three plant expression vectors expressing pre-amiRNAs were constructed, and recombinant plasmid DNAs (pDNAs) were loaded onto nontoxic and degradable layered double hydroxide (LDH) clay nanosheets. LDH nanosheets containing multiple pDNAs were sprayed onto plant leaves. We found that the designed amiRNAs were significantly accumulated in leaves 7 days after spraying, while the pDNAs were sustainably detected for 35 days after the spray, suggesting that the LDH nanosheets released pDNAs in a sustained manner, protected pDNAs from degradation and efficiently delivered pDNAs into plant cells. Importantly, when the LDH nanosheets coated with pDNAs were sprayed onto plants infected by TYLCV, both the disease severity and TYLCV viral concentration in sprayed plants were significantly decreased during the 35 days, while the levels of H2O2 were significantly increased in those plants. Taken together, these results indicate that LDH nanosheets loaded with pDNAs expressing amiRNAs can be a sustainable and promising tool for begomovirus control.

De Novo Assembly and Transcriptome Analysis of Wheat with Male Sterility Induced by the Chemical Hybridizing Agent SQ-1.

Wheat (Triticum aestivum L.), one of the world's most important food crops, is a strictly autogamous (self-pollinating) species with exclusively perfect flowers. Male sterility induced by chemical hybridizing agents has increasingly attracted attention as a tool for hybrid seed production in wheat; however, the molecular mechanisms of male sterility induced by the agent SQ-1 remain poorly understood due to limited whole transcriptome data. Therefore, a comparative analysis of wheat anther transcriptomes for male fertile wheat and SQ-1-induced male sterile wheat was carried out using next-generation sequencing technology. In all, 42,634,123 sequence reads were generated and were assembled into 82,356 high-quality unigenes with an average length of 724 bp. Of these, 1,088 unigenes were significantly differentially expressed in the fertile and sterile wheat anthers, including 643 up-regulated unigenes and 445 down-regulated unigenes. The differentially expressed unigenes with functional annotations were mapped onto 60 pathways using the Kyoto Encyclopedia of Genes and Genomes database. They were mainly involved in coding for the components of ribosomes, photosynthesis, respiration, purine and pyrimidine metabolism, amino acid metabolism, glutathione metabolism, RNA transport and signal transduction, reactive oxygen species metabolism, mRNA surveillance pathways, protein processing in the endoplasmic reticulum, protein export, and ubiquitin-mediated proteolysis. This study is the first to provide a systematic overview comparing wheat anther transcriptomes of male fertile wheat with those of SQ-1-induced male sterile wheat and is a valuable source of data for future research in SQ-1-induced wheat male sterility.