Construction of Host Plant Insect-Resistance Mutant Library by High-Throughput CRISPR/Cas9 System and Identification of A Broad-Spectrum Insect Resistance Gene.

Insects pose significant challenges in cotton-producing regions. Here, they describe a high-throughput CRISPR/Cas9-mediated large-scale mutagenesis library targeting endogenous insect-resistance-related genes in cotton. This library targeted 502 previously identified genes using 968 sgRNAs, generated ≈2000 T0 plants and achieved 97.29% genome editing with efficient heredity, reaching upto 84.78%. Several potential resistance-related mutants (10% of 200 lines) their identified that may contribute to cotton-insect molecular interaction. Among these, they selected 139 and 144 lines showing decreased resistance to pest infestation and targeting major latex-like protein 423 (GhMLP423) for in-depth study. Overexpression of GhMLP423 enhanced insect resistance by activating the plant systemic acquired resistance (SAR) of salicylic acid (SA) and pathogenesis-related (PR) genes. This activation is induced by an elevation of cytosolic calcium [Ca2+ ]cyt flux eliciting reactive oxygen species (ROS), which their demoted in GhMLP423 knockout (CR) plants. Protein-protein interaction assays revealed that GhMLP423 interacted with a human epidermal growth factor receptor substrate15 (EPS15) protein at the cell membrane. Together, they regulated the systemically propagating waves of Ca2+ and ROS, which in turn induced SAR. Collectively, this large-scale mutagenesis library provides an efficient strategy for functional genomics research of polyploid plant species and serves as a solid platform for genetic engineering of insect resistance.

The complex genome and adaptive evolution of polyploid Chinese pepper (Zanthoxylum armatum and Zanthoxylum bungeanum).

Zanthoxylum armatum and Zanthoxylum bungeanum, known as 'Chinese pepper', are distinguished by their extraordinary complex genomes, phenotypic innovation of adaptive evolution and species-special metabolites. Here, we report reference-grade genomes of Z. armatum and Z. bungeanum. Using high coverage sequence data and comprehensive assembly strategies, we derived 66 pseudochromosomes comprising 33 homologous phased groups of two subgenomes, including autotetraploid Z. armatum. The genomic rearrangements and two whole-genome duplications created large (~4.5 Gb) complex genomes with a high ratio of repetitive sequences (>82%) and high chromosome number (2n = 4x = 132). Further analysis of the high-quality genomes shed lights on the genomic basis of involutional reproduction, allomones biosynthesis and adaptive evolution in Chinese pepper, revealing a high consistent relationship between genomic evolution, environmental factors and phenotypic innovation. Our study provides genomic resources and new insights for investigating diversification and phenotypic innovation in Chinese pepper, with broader implications for the protection of plants under severe environmental changes.

Transcriptome analysis provides insights into the molecular mechanism of GhSAMDC1 involving in rapid vegetative growth and early flowering in tobacco.

In previous study, ectopic expression of GhSAMDC1 improved vegetative growth and early flowering in tobacco, which had been explained through changes of polyamine content, polyamines and flowering relate genes expression. To further disclose the transcript changes of ectopic expression of GhSAMDC1 in tobacco, the leaves from wild type and two transgenic lines at seedling (30 days old), bolting (60 days old) and flowering (90 days old) stages were performed for transcriptome analysis. Compared to wild type, a total of 938 differentially expressed genes (DEGs) were found to be up- or down-regulated in the two transgenic plants. GO and KEGG analysis revealed that tobacco of wild-type and transgenic lines were controlled by a complex gene network, which regulated multiple metabolic pathways. Phytohormone detection indicate GhSAMDC1 affect endogenous phytohormone content, ABA and JA content are remarkably increased in transgenic plants. Furthermore, transcript factor analysis indicated 18 transcript factor families, including stress response, development and flowering related transcript factor families, especially AP2-EREBP, WRKY, HSF and Tify are the most over-represented in those transcript factor families. In conclusion, transcriptome analysis provides insights into the molecular mechanism of GhSAMDC1 involving rapid vegetative growth and early flowering in tobacco.

Chromosome-level genome of Camellia lanceoleosa provides a valuable resource for understanding genome evolution and self-incompatibility.

The section Oleifera (Theaceae) has attracted attention for the high levels of unsaturated fatty acids found in its seeds. Here, we report the chromosome-scale genome of the sect. Oleifera using diploid wild Camellia lanceoleosa with a final size of 3.00 Gb and an N50 scaffold size of 186.43 Mb. Repetitive sequences accounted for 80.63% and were distributed unevenly across the genome. Camellia lanceoleosa underwent a whole-genome duplication event approximately 65 million years ago (65 Mya), prior to the divergence of C. lanceoleosa and Camellia sinensis (approx. 6-7 Mya). Syntenic comparisons of these two species elucidated the genomic rearrangement, appearing to be driven in part by the activity of transposable elements. The expanded and positively selected genes in C. lanceoleosa were significantly enriched in oil biosynthesis, and the expansion of homomeric acetyl-coenzyme A carboxylase (ACCase) genes and the seed-biased expression of genes encoding heteromeric ACCase, diacylglycerol acyltransferase, glyceraldehyde-3-phosphate dehydrogenase and stearoyl-ACP desaturase could be of primary importance for the high oil and oleic acid content found in C. lanceoleosa. Theanine and catechins were present in the leaves of C. lanceoleosa. However, caffeine can not be dectected in the leaves but was abundant in the seeds and roots. The functional and transcriptional divergence of genes encoding SAM-dependent N-methyltransferases may be associated with caffeine accumulation and distribution. Gene expression profiles, structural composition and chromosomal location suggest that the late-acting self-incompatibility of C. lanceoleosa is likely to have favoured a novel mechanism co-occurring with gametophytic self-incompatibility. This study provides valuable resources for quantitative and qualitative improvements and genome assembly of polyploid plants in sect. Oleifera.

Ectopic Expression of GhSAMDC3 Enhanced Salt Tolerance Due to Accumulated Spd Content and Activation of Salt Tolerance-Related Genes in Arabidopsis thaliana.

Polyamines (PAs), especially spermidine and spermine (which are involved in various types of abiotic stress tolerance), have been reported in many plant species. In this study, we identified 14 putative S-adenosylmethionine decarboxylase genes (GhSAMDC1-14) in upland cotton. Based on phylogenetic and expression analyses conducted under different abiotic stresses, we selected and transferred GhSAMDC3 into Arabidopsis thaliana. Compared to the wild type, transgenic plants displayed rapid growth and increases in average leaf area and leaf number of 52% and 36%, respectively. In transgenic plants, the germination vigor and rate were markedly enhanced under NaCl treatment, and the plant survival rate increased by 50% under 300 mM NaCl treatment. The spermidine content was significantly increased, possibly due to the synthesis of a series of PAs and oxidant and antioxidant genes, resulting in improved salinity tolerance in Arabidopsis. Various salinity resistance-related genes were upregulated in transgenic plants. Together, these results indicate that ectopic expression of GhSAMDC3 raised salinity tolerance by the accumulation of spermidine and activation of salinity tolerance-related genes in A. thaliana.

Ectopic expression of GhSAMDC1 improved plant vegetative growth and early flowering through conversion of spermidine to spermine in tobacco.

Polyamines play essential roles in plant development and various stress responses. In this study, one of the cotton S-adenosylmethionine decarboxylase (SAMDC) genes, GhSAMDC1, was constructed in the pGWB17 vector and overexpressed in tobacco. Leaf area and plant height increased 25.9-36.6% and 15.0-27.0%, respectively, compared to the wild type, and flowering time was advanced by 5 days in transgenic tobacco lines. Polyamine and gene expression analyses demonstrated that a decrease in spermidine and an increase in total polyamines and spermine might be regulated by NtSPDS4 and NtSPMS in transgenic plants. Furthermore, exogenous spermidine, spermine and spermidine synthesis inhibitor dicyclohexylamine were used for complementary tests, which resulted in small leaves and dwarf plants, big leaves and early flowering, and big leaves and dwarf plants, respectively. These results indicate that spermidine and spermine are mainly involved in the vegetative growth and early flowering stages, respectively. Expression analysis of flowering-related genes suggested that NtSOC1, NtAP1, NtNFL1 and NtFT4 were upregulated in transgenic plants. In conclusion, ectopic GhSAMDC1 is involved in the conversion of spermidine to spermine, resulting in rapid vegetative growth and early flowering in tobacco, which could be applied to genetically improve plants.

Simultaneous silencing of GhFAD2-1 and GhFATB enhances the quality of cottonseed oil with high oleic acid.

Cottonseed oil has become an important source of edible oil due to its significant cost advantage. However, there is a growing concern over its fatty acid composition and nutritional value. In Gossypium hirsutum, GhFAD2-1 and GhFATB encoding the microsomal oleate desaturase and palmitoyl-acyl carrier protein thioesterase, respectively, play critical roles in regulating the proportions of saturated and polyunsaturated fatty acids in cottonseed lipids. In this study, RNAi technology was used to simultaneously inhibit the expression levels of GhFAD2-1 and GhFATB to improve the quality of cottonseed oil by increasing oleic acid content. Transgenic cotton plants with reduced levels of both target genes were successfully generated. In mature seed kernels of transgenic plants, the content of oleic acid was 38.25%, accordingly increasing by 156.96%, while the content of palmitic acid and linoleic acid was 19.15% and 36.68%, decreasing by 21.28% and 33.92%, respectively, compared with that of the control. The total oil content in transgenic and control kernels was 22.48% and 29.83%, respectively. The reduced oil level in transgenic seeds was accompanied by a reduction in seed index, thereby causing disadvantageous effects on seed germination potentiality and seed vigor, particularly under cool stress conditions. Our results demonstrated the feasibility of simultaneous manipulation of multiple genes using RNAi technology and showed the important role of oil content in seed development and vigor. Our findings provide insight into the physiological significance of the fatty acid composition in cottonseeds.