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Cotton plants produce two classes of terpenoid defence compounds against pathogens and other pests. Both classes are derived from a common sesquiterpenoid precursor, δ‐cadinen‐2‐one, which enters either the gossypol pathway or the lacinilene pathway. Blocking the gossypol pathway by RNAi suppression of the early pathway biosynthetic enzyme CYP82D hydroxylase resulted in enhanced resistance to the Fusarium wilt pathogen. Analyses of root terpenoids revealed no overall increases in the products of the gossypol pathway in the roots infected by the wilt pathogen. However, the lacinilene pathway was elicited by the pathogen and the lacinilene levels were 19‐fold higher in the RNAi plants than in wild‐type plants. In the pathogen inoculated RNAi 73R plants, the concentrations of DHC and HMC were 231 μg and 886 μg/g dry roots, respectively, which may have contributed to the inhibition of fungal invasion. In comparison, the concentrations of DHC and HMC in the pathogen inoculated control wild‐type 73W plants were only 0.7 μg and 58 μg/g dry roots, respectively. Fungitoxicity testing showed that DHC at 100 μg/ml inhibited growth of the Fusarium wilt pathogen by >93%. Treatment with the phytohormone jasmonic acid failed to elicit production of lacinilene pathway terpenoids in roots of either RNAi plants or their wild‐type sibling lines, but increased production of gossypol pathway terpenoids with concentrations in RNAi plants 80%–97% less than those in wild‐type plants. This indicates that induction of the lacinilene pathway is not directly mediated by jasmonic acid signalling and requires other signalling to activate the pathway. These results illustrate possible mechanisms of wilt disease resistance in cotton and provide a new approach to increase host resistance by manipulating these two major cotton chemical defence pathways. [ABSTRACT FROM AUTHOR]

Summary: In seeds and other parts of cultivated, tetraploid cotton (Gossypium hirsutum L.), multicellular groups of cells lysigenously form dark glands containing toxic terpenoids such as gossypol that defend the plant against pests and pathogens. Using RNA‐seq analysis of embryos from near‐isogenic glanded (Gl2Gl2Gl3Gl3) versus glandless (gl2gl2gl3gl3) plants, we identified 33 genes that expressed exclusively or at higher levels in embryos just prior to gland formation in glanded plants. Virus‐induced gene silencing against three gene pairs led to significant reductions in the number of glands in the leaves, and significantly lower levels of gossypol and related terpenoids. These genes encode transcription factors and have been designated the 'Cotton Gland Formation' (CGF) genes. No sequence differences were found between glanded and glandless cotton for CGF1 and CGF2 gene pairs. The glandless cotton has a transposon insertion within the coding sequence of the GoPGF (synonym CGF3) gene of the A subgenome and extensive mutations in the promoter of D subgenome homeolog. Overexpression of GoPGF (synonym CGF3) led to a dramatic increase in gossypol and related terpenoids in cultured cells, whereas CRISPR/Cas9 knockout of GoPGF (synonym CGF3) genes resulted in glandless phenotype. Taken collectively, the results show that the GoPGF (synonym CGF3) gene plays a critical role in the formation of glands in the cotton plant. Seed‐specific silencing of CGF genes, either individually or in combination, could eliminate glands, thus gossypol, from the cottonseed to render it safe as food or feed for monogastrics. [ABSTRACT FROM AUTHOR]

Cottonseed remains a low-value by-product of lint production mainly due to the presence of toxic gossypol that makes it unfit for monogastrics. Ultra-low gossypol cottonseed (ULGCS) lines were developed using RNAi knockdown of δ-cadinene synthase gene(s) in Gossypium hirsutum. The purpose of the current study was to assess the stability and specificity of the ULGCS trait and evaluate the agronomic performance of the transgenic lines. Trials conducted over a period of 3 years show that the ULGCS trait was stable under field conditions and the foliage/floral organs of transgenic lines contained wild-type levels of gossypol and related terpenoids. Although it was a relatively small-scale study, we did not observe any negative effects on either the yield or quality of the fibre and seed in the transgenic lines compared with the nontransgenic parental plants. Compositional analysis was performed on the seeds obtained from plants grown in the field during 2009. As expected, the major difference between the ULGCS and wild-type cottonseeds was in terms of their gossypol levels. With the exception of oil content, the composition of ULGCS was similar to that of nontransgenic cottonseeds. Interestingly, the ULGCS had significantly higher (4%-8%) oil content compared with the seeds from the nontransgenic parent. Field trial results confirmed the stability and specificity of the ULGCS trait suggesting that this RNAi-based product has the potential to be commercially viable. Thus, it may be possible to enhance and expand the nutritional utility of the annual cottonseed output to fulfil the ever-increasing needs of humanity. [ABSTRACT FROM AUTHOR]

Summary Cottonseed, containing 22.5% protein, remains an under-utilized and under-valued resource because of the presence of toxic gossypol. RNAi-knockdown of δ-cadinene synthase gene(s) was used to engineer plants that produced ultra-low gossypol cottonseed (ULGCS). In the original study, we observed that RNAi plants, a month or older, maintain normal complement of gossypol and related terpenoids in the roots, foliage, floral organs, and young bolls. However, the terpenoid levels and profile of the RNAi lines during the early stages of germination, under normal conditions and in response to pathogen exposure, had not been examined. Results obtained in this study show that during the early stages of seed germination/seedling growth, in both non-transgenic and RNAi lines, the tissues derived directly from bulk of the seed kernel (cotyledon and hypocotyl) synthesize little, if any new terpenoids. However, the growing root tissue and the emerging true leaves of RNAi seedlings showed normal, wild-type terpenoid levels. Biochemical and molecular analyses showed that pathogen-challenged parts of RNAi seedlings are capable of launching a terpenoid-based defence response. Nine different RNAi lines were monitored for five generations. The results show that, unlike the unstable nature of antisense-mediated low seed-gossypol phenotype, the RNAi-mediated ULGCS trait exhibited multi-generational stability. [ABSTRACT FROM AUTHOR]

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