Cotton miR319b-Targeted TCP4-Like Enhances Plant Defense Against Verticillium dahliae by Activating GhICS1 Transcription Expression.

Teosinte branched1/Cincinnata/proliferating cell factor (TCP) transcription factors play important roles in plant growth and defense. However, the molecular mechanisms of TCPs participating in plant defense remain unclear. Here, we characterized a cotton TCP4-like fine-tuned by miR319b, which could interact with NON-EXPRESSER OF PATHOGEN-RELATED GENES 1 (NPR1) to directly activate isochorismate synthase 1 (ICS1) expression, facilitating plant resistance against Verticillium dahliae. mRNA degradome data and GUS-fused assay showed that GhTCP4-like mRNA was directedly cleaved by ghr-miR319b. Knockdown of ghr-miR319b increased plant resistance to V. dahliae, whereas silencing GhTCP4-like increased plant susceptibility by the virus-induced gene silencing (VIGS) method, suggesting that GhTCP4-like is a positive regulator of plant defense. According to the electrophoretic mobility shift assay and GUS reporter analysis, GhTCP4-like could transcriptionally activate GhICS1 expression, resulting in increased salicylic acid (SA) accumulation. Yeast two-hybrid and luciferase complementation image analyses demonstrated that GhTCP4-like interacts with GhNPR1, which can promote GhTCP4-like transcriptional activation in GhICS1 expression according to the GUS reporter assay. Together, these results revealed that GhTCP4-like interacts with GhNPR1 to promote GhICS1 expression through fine-tuning of ghr-miR319b, leading to SA accumulation, which is percepted by NPR1 to increase plant defense against V. dahliae. Therefore, GhTCP4-like participates in a positive feedback regulation loop of SA biosynthesis via NPR1, increasing plant defenses against fungal infection.

Genome sequence of Gossypium anomalum facilitates interspecific introgression breeding.

Crop wild relatives are an important reservoir of natural biodiversity. However, incorporating wild genetic diversity into breeding programs is often hampered by reproductive barriers and a lack of accurate genomic information. We assembled a high-quality, accurately centromere-anchored genome of Gossypium anomalum, a stress-tolerant wild cotton species. We provided a strategy to discover and transfer agronomically valuable genes from wild diploid species to tetraploid cotton cultivars. With a (Gossypium hirsutum × G. anomalum)2 hexaploid as a bridge parent, we developed a set of 74 diploid chromosome segment substitution lines (CSSLs) of the wild cotton species G. anomalum in the G. hirsutum background. This set of CSSLs included 70 homozygous substitutions and four heterozygous substitutions, and it collectively contained about 72.22% of the G. anomalum genome. Twenty-four quantitative trait loci associated with plant height, yield, and fiber qualities were detected on 15 substitution segments. Integrating the reference genome with agronomic trait evaluation of the CSSLs enabled location and cloning of two G. anomalum genes that encode peroxiredoxin and putative callose synthase 8, respectively, conferring drought tolerance and improving fiber strength. We have demonstrated the power of a high-quality wild-species reference genome for identifying agronomically valuable alleles to facilitate interspecific introgression breeding in crops.

A Cotton Lignin Biosynthesis Gene, GhLAC4, Fine-Tuned by ghr-miR397 Modulates Plant Resistance Against Verticillium dahliae.

Plant lignin is a component of the cell wall, and plays important roles in the transport potential of water and mineral nutrition and plant defence against biotic stresses. Therefore, it is necessary to identify lignin biosynthesis-related genes and dissect their functions and underlying mechanisms. Here, we characterised a cotton LAC, GhLAC4, which participates in lignin biosynthesis and plant resistance against Verticillium dahliae. According to degradome sequencing and GUS reporter analysis, ghr-miR397 was identified to directedly cleave the GhLAC4 transcript through base complementary. GhLAC4 knockdown and ghr-miR397 overexpression significantly reduced basal lignin content compared to the control, whereas ghr-miR397 silencing significantly increased basal lignin levels. Based on staining patterns and GC/MS analysis, GhLAC4 acted in G-lignin biosynthesis. Under V. dahliae infection, we found that G-lignin content in ghr-miR397-knockdowned plants significantly increased, compared to these plants under the mock treatment, while G-lignin contents in GhLAC4-silenced plants and ghr-miR397-overexpressed plants treated with pathogen were comparable with these plants treated with mock, indicating that GhLAC4 participates in defence-induced G-lignin biosynthesis in the cell wall. Knockdown of ghr-miR397 in plants inoculated with V. dahliae promoted lignin accumulation and increased plant resistance. The overexpression of ghr-miR397 and knockdown of GhLAC4 reduced lignin content and showed higher susceptibility of plants to the fungal infection compared to the control. The extract-free stems of ghr-miR397-knockdowned plants lost significantly less weight when treated with commercial cellulase and V. dahliae secretion compared to the control, while the stems of ghr-miR397-overexpressed and GhLAC4-silenced plants showed significantly higher loss of weight. These results suggest that lignin protects plant cell walls from degradation mediated by cellulase or fungal secretions. In summary, the ghr-miR397-GhLAC4 module regulates both basal lignin and defence-induced lignin biosynthesis and increases plant resistance against infection by V. dahliae.