Prof. Youjun Zhang and his collaborators have devoted nearly 20 years of research to revealing the mechanisms of Bt resistance in the diamondback moth, Plutella xylostella (L.). Previously, they had demonstrated that the insect hormone-activated MAPK signalling pathway conferring Bt resistance to the diamondback moth by trans-regulating the differential expression of multiple midgut genes, including receptor and non-receptor paralogous genes . This report represents a great breakthrough in understanding the underlying transcriptional control mechanisms of this response.
Le Guo joined the lab in 2015 and was assigned to engage in the transcriptional regulatory mechanism of Bt resistance, and Jianying Qin joined us in 2017. During a seminar in our lab, Prof. Zhaojiang Guo queries how the MAPK signalling pathway orchestrated the differential expression of multiple genes and hypothesised that this might be regulated by transcription factors (TFs). Through series of works, we revealed two TFs, GATAd and Jun that could regulate the expression of ALP  and ABCB1 , respectively, while the regulatory mechanisms of the other receptors (APN1, APN3a, ABCC2, ABCC3) and non-receptors (APN5, APN6 and ABCC1) remained unresolved.
We started by cloning the promoter region of all relevant midgut genes, but found it extremely difficult to identify potential TFs. However, we read about a single transcription factor which promoted both yield and immunity in rice and realized that all or most of our midgut genes may also be regulated by one TF. Since we know that MAPK cascades typically activate downstream TFs via phosphorylation, Jianying Qin analyzed our previous phosphoproteomics assays from Bt Cry1Ac susceptible and resistant strains , and identified several transcription factors with differential levels of phosphorylation.
One of these, a TF called fushi tarazu factor 1 (FTZ-F1), was preliminarily shown to have regulatory activity on all of our target genes. This was an exciting finding but there was a problem in that we couldn’t identify consistent binding sites in our gene promoters, but that didn't stop us from moving forward. The phosphoproteomics data showed that the FTZ-F1 phosphorylation was increased in the resistant strain, so we hypothesized that phosphorylation may influence the regulatory role of FTZ-F1.
What then followed was about a year of exploration as to how to identify functional phosphorylation sites and to detect the phosphorylation level of FTZ-F1. Fan Ye joined the project and optimized an experimental system for detecting changes in protein phosphorylation level. Finally, our efforts demonstrated that the phosphorylation level of FTZ-F1 was modulated by the MAPK signaling pathway. Meanwhile, Dan Sun performed CRIPSR/Cas9 knock outs of all non-receptor genes from the resistant strain to provide experimental confirmation that these genes could offset the fitness costs resulting from the downregulation of the physiologically important receptor proteins.
Having obtained these nice data we then faced the daunting task of putting together a complex manuscript and are thankful for the assistance of our international project advisors for providing advice on how best to present the data and for proofreading early drafts. Finally, we have successfully published this five-year study in Nature Communications .
- Guo Z, Kang S, Chen D, Wu Q, Wang S, Xie W, Zhu X, Baxter SW, Zhou X, Jurat-Fuentes JL, Zhang Y. MAPK signaling pathway alters expression of midgut ALP and ABCC genes and causes resistance to Bacillus thuringiensis Cry1Ac toxin in diamondback moth. PLoS Genet., 2015, 11(4): e1005124.
- Guo Z, Kang S, Sun D, Gong L, Zhou J, Qin J, Guo L, Zhu L, Bai Y, Ye F, Wu Q, Wang S, Crickmore N, Zhou X, Zhang Y. MAPK-dependent hormonal signaling plasticity contributes to overcoming Bacillus thuringiensis toxin action in an insect host. Nat. Commun., 2020, 11(1): 3003.
- Guo Z, Kang S, Wu Q, Wang S, Crickmore N, Zhou X, Bravo A, Soberón M, Zhang Y. The regulation landscape of MAPK signaling cascade for thwarting Bacillus thuringiensis infection in an insect host. PLoS Pathog., 2021, 17(9): e1009917.
- Sun D, Zhu L, Guo L, Wang S, Wu Q, Crickmore N, Zhou X, Bravo A, Soberón M, Guo Z, Zhang Y. A versatile contribution of both aminopeptidases N and ABC transporters to Bt Cry1Ac toxicity in the diamondback moth. BMC Biol., 2022, 20(1): 33.
- Guo L, Cheng Z, Qin J, Sun D, Wang S, Wu Q, Crickmore N, Zhou X, Bravo A, Soberón M, Guo Z, Zhang Y. MAPK-mediated transcription factor GATAd contributes to Cry1Ac resistance in diamondback moth by reducing PxmALP expression. PLoS Genet., 2022, 18(2): e1010037.
- Qin J, Guo L, Ye F, Kang S, Sun D, Zhu L, Bai Y, Cheng Z, Xu L, Ouyang C, Xiao L, Wang S, Wu Q, Zhou X, Crickmore N, Zhou X, Guo Z, Zhang Y. MAPK-activated transcription factor PxJun suppresses PxABCB1 expression and confers resistance to Bacillus thuringiensis Cry1Ac toxin in Plutella xylostella (L.). Appl. Environ. Microbiol., 2021, 87(13): e00466-21.
- Guo Z, Guo L, Qin J, Ye F, Sun D, Wu Q, Wang S, Crickmore N, Zhou X, Bravo A, Soberón M, Zhang Y. A single transcription factor facilitates an insect host combating Bacillus thuringiensis infection while maintaining fitness. Nat. Commun., 2022, 13(1): 6024.