The temperature and light-dependent molecular dynamic investigation on foxtail millet’s blast effector APikL2A/sHMA25

The temperature and light-dependent molecular dynamic investigation on foxtail millet’s blast effector APikL2A/sHMA25 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The temperature and light-dependent molecular dynamic investigation on foxtail millet’s blast effector APikL2A/sHMA25 Ling Zhao, Ting Zhang, Yanjie Luo, Lin Li, Ruhong Cheng, Zhigang Shi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1576885/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 6 You are reading this latest preprint version Abstract Magnaporthe oryzae is the causal agent of rice blast. Understanding the abiotic stress influence on the resistance possess is benefit to regulate the disease. Here, effects of thermal and light (oscillating electric field) on the complex of Magnaporthe oryzae effector APikL2A/sHMA25 from foxtail millet had been investigated by molecular dynamic (MD) simulations using Gromacs package. The structure of APikL2A/sHMA25 were relatively stable within a temperature range from 290 K (16.85°C) to 320 K (46.85°C), and a slightly higher surface area was observed at higher temperature. In contrary, we found that the oscillating electric fields had a notable influence on the structure of APikL2A/sHMA25, and destructive effects could be detected at a field intensity about 0.5 V/nm. Alpha-helix structures were the least stable under oscillating electric fields, because of the existence of their dipole moment, while beta-sheet was quite be stable in the oscillating electric fields. blast effector molecular dynamic simulations temperature oscillating electric field Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction The blast disease caused by the fungal pathogen Magnaporthe oryzae is one of the most limiting factors to crop production worldwide. This disease could infect at least 59 species of grasses, such as rice, wheat, maize, barley, foxtail millet, and their wild relatives, which result in serious yield losses in crop harvests and thus threaten the global food security [ 1 – 3 ]. For rice, the blast disease could lead to a yield loss up to 100% in infected region and a global yield loss that enough to feed 212–742 million people yearly [ 3 , 4 ]. It drove people insight into the mechanism of pathogenesis to develop effective methods to control the disease. As a hemibiotroph pathogen, M. oryzae initial the infection by a biotrophic interaction with the host[ 5 ]. During the infection processes, the effector proteins were secreted by M. oryzae and interacted with the host plants, thus inhibiting their immune responses and changing the metabolism [ 6 , 7 ]. On the other hand, the intracellular immune receptors of host plants could recognize the effectors and remarkable trigger the immune responses to resist the attacks [ 8 ]. Understanding the function of M. oryzae effectors, their host targets, and pathogen avirulent (AVR)-resistance (R) gene interactions are the key factors to elaborate the mechanism of M. oryzae infection. To date, numerous R genes (Pb1, Pia, Pib, Pid2, Pid3, Pik, Pikh/Pi54, Pikm, Pikp, Pish, Pit, Pita, Pizt, Pi1, Pi2, Pi5, Pi9, pi21, Pi25, Pi36, Pi37, Pi56, Pi63, PiCO39, Pi64, Pigm)and AVR genes (AVR-Pi54, AVR-Pi9, AVR-Pib, AVR-Pia, AVR-Pii, AVR-Pik/km/kp, AVR-Pizt, ACE1, AVR-Pita, AVR1-CO39, PWL1, PWL2) had been cloned [ 9 – 21 ]. Among these genes, AVR-Pik effectors had been widely investigated. Maidment et al. demonstrated that AVR-PikD could interact with both the heavy-metal-associated (HMA) domain of OsHIPP19 and the integrated HMA domains of Pik-1 alleles, while the former interaction was tighter [ 22 ]. Maqbool et al. showed the crystal structure of Pikp-HMA/AVR-PikD complex and detailed the immune recognition event in tobacco ( Nicotiana benthamiana ) [ 1 ]. In addition, the crystal structure of effector proteins APikL2A (with host target sHMA25) and APikL2F (with host target sHMA94) from foxtail millet had been revealed by X-ray diffraction with a resolution better than 2.3 Å [ 23 ]. It is well known that changes in temperatures, light and other conditions could significantly affect the pathogen infections. Rajput et al. found that at supraoptimal (32°C) appressoria formation and spore germination for M. oryzae were much slower than that at the optimal temperature (27°C), which suggested M. oryzae may be noninfectious at higher temperature [ 24 ]. Qiu et al. demonstrated that jasmonic acid biosynthesis and signaling genes in rice could be effectively induced by M. oryzae at 28°C but not at 22°C [ 25 ]. Madhusudhan et al. observed lower expression of Pi54 and WRKY45 at the optimal temperature for pathogen aggressiveness which indicated that possible temoerature-induced interruption of the defence prcesses [ 26 ]. In addition to temperature, light is another factor that could affect fungal infection. Lee at al. observed that both blue and red light could suppress the spore-release of M. oryzae , while its asexual development only suppressed by blue light [ 27 ]. In addition, Li et al. also indicated that ultraviolet radiation could weaken the infectivity of M. oryzae [ 28 ]. However, the effects of temperature and light on M. oryzae effector proteins are still unclear. Molecular dynamic (MD) simulation is a powerful tool for studying biomolecular structure and dynamics in a manner similar to the experiments [ 29 ]. To quantify the effects of temperature and light on the dynamics of effector proteins, here we performed MD simulations on the complex of APikL2A/sHMA25. The root mean square deviation (RMSD), radius of gyration ( R g ), surface areas and secondary structure at different temperature and light conditions were presented. While the increasing of temperature from 290 K (16.85°C) to 320 K (46.85°C) had a slight influence on the structure of APikL2A/sHMA25, the strong light (oscillating electric field) could remarkably damage the protein structure, especially for the alpha-helix structures. 2. Methods MD simulations were conducted by the Gromacs package with the CHARMM36 force field and SPCE water model [ 30 , 31 ]. The structure of APikL2A/sHMA25 was obtained from protein data bank with the PDB ID 7NLJ [ 23 ]. This protein was placed at the center of a cubic water box with a minimum distance of 1 nm to the box edge, and the system was neutralized by adding 6 Cl - ions. Then, the energy minimization, NVT and NPT equilibration, 10 ns MD simulations were performed at four selected temperatures, that is 290 K (16.85°C), 300 K (26.85°C), 310 K (36.85°C) and 320 K (46.85°C) for temperature-dependent simulations. The monochromatic light corresponding to a wavelength ~ 400 nm realized by oscillating electric field[ 32 ], $$E\left(t\right)={E}_{0}\text{e}\text{x}\text{p}[-\frac{{\left(t-{t}_{0}\right)}^{2}}{2{\sigma }^{2}}]\text{c}\text{o}\text{s}\left[{\omega }\left(t-{t}_{0}\right)\right]$$ where ω = 2πc/λ; σ = 0 (for continuous wave), and E 0 is set to be 0.2, 0.4, 0.5 and 0.6 V/nm. In addition, the analyzation of dynamic data was conducted by Gromacs tools unless otherwise specified. 3. Results 3.1. Thermal stresses The convergence of MD simulations was checked by temperature, pressure, density, potential energy and other thermodynamic parameters. All of these parameters indicated that the system converged within 10 ns simulations. The density and potential energy did not change dramatically at different temperature. As the temperature increasing from 290 K to 320 K, the volumes increased from 403.60 to 410.23 nm 3 and the densities decreased from 1024.01 to 1007.39 kg/m 3 . The potential energy was − 622591 kJ/mol, -614210 kJ/mol, -606173 kJ/mol and − 598067 kJ/mol for 290 K, 300 K, 310 K and 320 K, respectively. Figure 1 a showed the RMSD of backbone atoms. Although the RMSD of 310 K and 320 K had more visible fluctuations than the RMSD of lower temperature, the value of RMSD remained at ~ 0.15 nm. The time-dependent curves indicated that structures became relatively stable within 10 ns for all selected temperature. The calculated radius of gyration ( R g ) was shown in Fig. 1 b for different temperatures. Generally, each R g seemed time-independent with a typical constant around 1.62 nm. However, their values increased slightly with a rise in temperature, indicating structural changes might occur at high temperature. To obtain surface areas, the gmx_sasa command in Gromacs were performed [ 33 ], and the time-dependent surface information for different temperature were given in Fig. 1 c. It could be seen that the surface area increased with the rise of temperature. At 10 ns, the surface areas were 94.68 nm 2 , 94.90 nm 2 , 95.90 nm 2 and 95.15 nm 2 for 290 K, 300 K, 310 K and 320 K, respectively. Dictionary of protein secondary structure (DSSP) was a standard tool for the annotation of secondary structure elements from protein structures. APikL2A together with its host sHMA25 exhibited 169 residues [ 23 ]. The main components of these residues were beta-sheet (~ 39%), coil (~ 24%), alpha-helix (~ 18%). Figure 3 showed the evolution of time-dependent secondary structure for the selected temperatures. It could be seen that the secondary structure tended to be unchanged during the MD simulations, except some unstable features observed for alpha-helix structure at 310 K. 3.2. Oscillating electric field stresses The RMSD, Rg and surface area for different oscillating electric fields at 300 K were given in Fig. 3 . For 0.2 V/nm and 0.4 V/nm, their time-dependent behaviors were similar to the behaviors without electric fields and could converge in 10 ns, while the time-dependent behaviors of 0.5 V/nm and 0.6 V/nm seemed not converged and presented large values. Table 1 showed the average of RMSD, R g and surface area within 8–10 ns. All of them increase with the increasing of electric field. In particular, there was a sudden increase at 0.5 V/nm, indicating the dramatically structure changes occurred. To further elaborate the changes of structure, DSSP patterns were given in Fig. 4 . No significantly secondary structure changes observed for 0.2 V/nm and 0.4 V/nm. However, when E 0 = 0.5 and 0.6 V/nm, the alpha-helix structure became unstable, especially for the residues around 85 and 60. Further increasing electric field, i.e. 1 V/nm, could destroy all secondary structures, and we also found that beta-sheet and bend structure were more stable than the other structures. Table 1 The RMSD, R g and surface area for different oscillating electric fields within 8–10 ns. Field intensity (V/nm) 0 0.2 0.4 0.5 0.6 RMSD (nm) 0.1146 0.1136 0.1927 0.5587 0.2755 R g (nm) 1.6042 1.6195 1.6156 1.6505 1.6531 Surface area (nm 2 ) 94.5727 96.5900 96.9242 104.4414 105.1013 4. Discussion The abiotic stresses play important roles in the pathogen infection. By performing MD simulations, we exhibited the thermal and electric field (light)-dependent structural changes of APikL2A/sHMA25, a complex of rice blast effector protein with its host target from foxtail millet. All the results indicated that the structure of APikL2A/sHMA25 were relatively stable within a temperature range from 290 K to 320 K. In addition, we noticed a slightly higher surface area at higher temperature. Such an increased surface area might affect the reaction efficiency, since many protein interactions, e.g., protein-ligand binding, ion transport, and protein folding, occured at the interface between the protein and the solvent. On the other hand, the RMSD, Rg and surface area were significantly increased under the oscillating electric fields. This might be because of the oscillating field forced the amino acid residues to move in the direction of the field [ 34 ]. Further increasing of the electric field intensity might destroy the crystal structure of protein. In APikL2A/sHMA25, this easily occurred for the residues around 85 and 60 with an alpha-helix structure, because of alpha-helixes carrying their own dipole moment, thus align themself under the oscillating electric fields. In addition, we pointed out that the beta-sheet may be the most stable in the oscillating electric fields. Declarations Funding: This work was supported by National Key R&D program of China (2018YFD1000705); the National Natural Science Foundation of China (32101815); HAAFS Agriculture Science and Technology Innovation Project (2022KJCXZX-GZS-1); China Agricultural Research System (CARS-06-14.5); Hebei Agricultural Research System (HBCT2018070101, HBCT2018070201) and Hebei Seed Industry Innovation Special Project(21326302D) and Hebei Province Talent Plan project (A202103001, C20210509); Talents construction project of science and technology innovation, Hebei Academy of Agriculture and Forestry Science (C22R0402). Conflicts of interest: The authors have no relevant financial or non-financial interests to disclose. Availability of data and material: Further data sets are available from the corresponding author on reasonable. Code availability: GROMACS Software has been used under the GNU Lesser General Public License. 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Innov Food Sci Emerg 35:9–20. https://doi.org/10.1016/j.ifset.2016.03.004 Supplementary Files GA.pdf Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Major revisions needed 21 Aug, 2022 Reviewers agreed at journal 30 Jul, 2022 Reviewers invited by journal 01 Jul, 2022 Editor invited by journal 21 Apr, 2022 Editor assigned by journal 21 Apr, 2022 First submitted to journal 20 Apr, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1576885","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":117811365,"identity":"d45e34eb-b95a-4752-b873-62a6df6fda4e","order_by":0,"name":"Ling Zhao","email":"","orcid":"","institution":"Hebei Academy of Agriculture and Forestry Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"Zhao","suffix":""},{"id":117811366,"identity":"4755cb48-00ee-4777-b8d0-89d3d9b4132b","order_by":1,"name":"Ting Zhang","email":"","orcid":"","institution":"Hebei Academy of Agriculture and Forestry Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Zhang","suffix":""},{"id":117811367,"identity":"9236fd74-c58b-4eac-b812-b6d5e7767a23","order_by":2,"name":"Yanjie Luo","email":"","orcid":"","institution":"Hebei Academy of Agriculture and Forestry Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yanjie","middleName":"","lastName":"Luo","suffix":""},{"id":117811368,"identity":"5958dd66-cfd5-4ffa-9e7e-01b149dda56e","order_by":3,"name":"Lin Li","email":"","orcid":"","institution":"Hebei Academy of Agriculture and Forestry Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Li","suffix":""},{"id":117811369,"identity":"28d3f6ba-89b9-4207-98cd-cdfd028e7263","order_by":4,"name":"Ruhong Cheng","email":"","orcid":"","institution":"Hebei Academy of Agriculture and Forestry Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ruhong","middleName":"","lastName":"Cheng","suffix":""},{"id":117811370,"identity":"bc1c0ce7-e202-4510-a9f0-2a2faea0f1b4","order_by":5,"name":"Zhigang Shi","email":"","orcid":"","institution":"Hebei Academy of Agriculture and Forestry Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhigang","middleName":"","lastName":"Shi","suffix":""},{"id":117811371,"identity":"50a280c2-2d74-4dd2-abd0-37a848522844","order_by":6,"name":"Genping Wang","email":"","orcid":"","institution":"Hebei Academy of Agriculture and Forestry Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Genping","middleName":"","lastName":"Wang","suffix":""},{"id":117811372,"identity":"90d8c146-303a-4a10-ae61-565281574ffd","order_by":7,"name":"Tiancong Ren","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYJCCAx8qJOTY2JsPEKmejYHx4IwzFsZ8PMcSiNbCfJi3rSJxnkSOAnE6zOc3PwBqkUhvY8hhYPhRsY2wFpljbAYH55yTyG1jOHuAsefMbcJaJNh4GA68KQNqYexLYGZsI1YLD5tEOhszjwHxWg7ytEkksLERryXNABjIEoZtPGwJB4nzC/Phxx8+VNTJy89/fPDBjwoitKCAAySqHwWjYBSMglGACwAAVnc4uOBbS2UAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-1272-4554","institution":"Shijiazhuang University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Tiancong","middleName":"","lastName":"Ren","suffix":""}],"badges":[],"createdAt":"2022-04-20 13:47:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1576885/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1576885/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":23640286,"identity":"200903fa-96f5-403c-97cb-ae53822efaee","added_by":"auto","created_at":"2022-07-08 16:18:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":307968,"visible":true,"origin":"","legend":"\u003cp\u003eThe MD results for 290 K, 300 K, 310 K and 320 K. (a) RMSD; (b) Rg; (c) Surface areas.\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-1576885/v1/896915426c317fb7b4ab1ce9.png"},{"id":23640285,"identity":"c903cac8-4233-4aba-ade9-b955db64e061","added_by":"auto","created_at":"2022-07-08 16:18:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1989655,"visible":true,"origin":"","legend":"\u003cp\u003eDictionary of protein secondary structure for different temperature. (a) 290 K; (b) 300 K; (c) 310 K; (d) 320 K.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-1576885/v1/c2a56682649aca4a3478b39a.png"},{"id":23640704,"identity":"3c61dda0-05fa-4008-a909-59bdd11aef5d","added_by":"auto","created_at":"2022-07-08 16:23:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":292880,"visible":true,"origin":"","legend":"\u003cp\u003eThe MD results for different oscillating electric fields at 300 K. (a) RMSD; (b) Rg; (c) Surface areas.\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-1576885/v1/b637ad91a2065b7ff6661894.png"},{"id":23640284,"identity":"f47c412a-6ad2-4c65-a6b8-aa197dbe1586","added_by":"auto","created_at":"2022-07-08 16:18:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1975998,"visible":true,"origin":"","legend":"\u003cp\u003eDictionary of protein secondary structure for different oscillating electric fields at 300 K. (a) 0.2 V/nm; (b) 0.4 V/nm K; (c) 0.5 V/nm K; (d) 0.6 V/nm.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-1576885/v1/d48c57702d3c03a0bc5df244.png"},{"id":23640707,"identity":"269985dd-bc8e-4a42-92b5-da8f1443860d","added_by":"auto","created_at":"2022-07-08 16:23:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":240779,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1576885/v1/c37f7204-0b46-4059-8954-1d94c516aeb5.pdf"},{"id":23640288,"identity":"e559da48-9266-43b8-8d18-9ccf89c7d6b8","added_by":"auto","created_at":"2022-07-08 16:18:44","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":959619,"visible":true,"origin":"","legend":"","description":"","filename":"GA.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1576885/v1/f10fafce48a0956ba134c9c7.pdf"}],"financialInterests":"","formattedTitle":"The temperature and light-dependent molecular dynamic investigation on foxtail millet’s blast effector APikL2A/sHMA25","fulltext":[{"header":"1. Introduction","content":"\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe blast disease caused by the fungal pathogen \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e is one of the most limiting factors to crop production worldwide. This disease could infect at least 59 species of grasses, such as rice, wheat, maize, barley, foxtail millet, and their wild relatives, which result in serious yield losses in crop harvests and thus threaten the global food security [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]. For rice, the blast disease could lead to a yield loss up to 100% in infected region and a global yield loss that enough to feed 212\u0026ndash;742\u0026nbsp;million people yearly [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. It drove people insight into the mechanism of pathogenesis to develop effective methods to control the disease.\u003c/p\u003e\n \u003cp\u003eAs a hemibiotroph pathogen, \u003cem\u003eM. oryzae\u003c/em\u003e initial the infection by a biotrophic interaction with the host[\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]. During the infection processes, the effector proteins were secreted by \u003cem\u003eM. oryzae\u003c/em\u003e and interacted with the host plants, thus inhibiting their immune responses and changing the metabolism [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. On the other hand, the intracellular immune receptors of host plants could recognize the effectors and remarkable trigger the immune responses to resist the attacks [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. Understanding the function of \u003cem\u003eM. oryzae\u003c/em\u003e effectors, their host targets, and pathogen avirulent (AVR)-resistance (R) gene interactions are the key factors to elaborate the mechanism of \u003cem\u003eM. oryzae\u003c/em\u003e infection.\u003c/p\u003e\n \u003cp\u003eTo date, numerous R genes (Pb1, Pia, Pib, Pid2, Pid3, Pik, Pikh/Pi54, Pikm, Pikp, Pish, Pit, Pita, Pizt, Pi1, Pi2, Pi5, Pi9, pi21, Pi25, Pi36, Pi37, Pi56, Pi63, PiCO39, Pi64, Pigm)and AVR genes (AVR-Pi54, AVR-Pi9, AVR-Pib, AVR-Pia, AVR-Pii, AVR-Pik/km/kp, AVR-Pizt, ACE1, AVR-Pita, AVR1-CO39, PWL1, PWL2) had been cloned [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. Among these genes, AVR-Pik effectors had been widely investigated. Maidment \u003cem\u003eet al.\u003c/em\u003e demonstrated that AVR-PikD could interact with both the heavy-metal-associated (HMA) domain of OsHIPP19 and the integrated HMA domains of Pik-1 alleles, while the former interaction was tighter [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. Maqbool \u003cem\u003eet al.\u003c/em\u003e showed the crystal structure of Pikp-HMA/AVR-PikD complex and detailed the immune recognition event in tobacco (\u003cem\u003eNicotiana benthamiana\u003c/em\u003e) [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. In addition, the crystal structure of effector proteins APikL2A (with host target sHMA25) and APikL2F (with host target sHMA94) from foxtail millet had been revealed by X-ray diffraction with a resolution better than 2.3 \u0026Aring; [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eIt is well known that changes in temperatures, light and other conditions could significantly affect the pathogen infections. Rajput \u003cem\u003eet al.\u003c/em\u003e found that at supraoptimal (32\u0026deg;C) appressoria formation and spore germination for \u003cem\u003eM. oryzae\u003c/em\u003e were much slower than that at the optimal temperature (27\u0026deg;C), which suggested \u003cem\u003eM. oryzae\u003c/em\u003e may be noninfectious at higher temperature [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. Qiu \u003cem\u003eet al.\u003c/em\u003e demonstrated that jasmonic acid biosynthesis and signaling genes in rice could be effectively induced by \u003cem\u003eM. oryzae\u003c/em\u003e at 28\u0026deg;C but not at 22\u0026deg;C [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. Madhusudhan \u003cem\u003eet al.\u003c/em\u003e observed lower expression of Pi54 and WRKY45 at the optimal temperature for pathogen aggressiveness which indicated that possible temoerature-induced interruption of the defence prcesses [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. In addition to temperature, light is another factor that could affect fungal infection. Lee \u003cem\u003eat al.\u003c/em\u003e observed that both blue and red light could suppress the spore-release of \u003cem\u003eM. oryzae\u003c/em\u003e, while its asexual development only suppressed by blue light [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. In addition, Li \u003cem\u003eet al.\u003c/em\u003e also indicated that ultraviolet radiation could weaken the infectivity of \u003cem\u003eM. oryzae\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, the effects of temperature and light on \u003cem\u003eM. oryzae\u003c/em\u003e effector proteins are still unclear.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eMolecular dynamic (MD) simulation is a powerful tool for studying biomolecular structure and dynamics in a manner similar to the experiments [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. To quantify the effects of temperature and light on the dynamics of effector proteins, here we performed MD simulations on the complex of APikL2A/sHMA25. The root mean square deviation (RMSD), radius of gyration (\u003cem\u003eR\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e), surface areas and secondary structure at different temperature and light conditions were presented. While the increasing of temperature from 290 K (16.85\u0026deg;C) to 320 K (46.85\u0026deg;C) had a slight influence on the structure of APikL2A/sHMA25, the strong light (oscillating electric field) could remarkably damage the protein structure, especially for the alpha-helix structures.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMD simulations were conducted by the Gromacs package with the CHARMM36 force field and SPCE water model [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The structure of APikL2A/sHMA25 was obtained from protein data bank with the PDB ID 7NLJ [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This protein was placed at the center of a cubic water box with a minimum distance of 1 nm to the box edge, and the system was neutralized by adding 6 Cl\u003csup\u003e-\u003c/sup\u003e ions. Then, the energy minimization, NVT and NPT equilibration, 10 ns MD simulations were performed at four selected temperatures, that is 290 K (16.85\u0026deg;C), 300 K (26.85\u0026deg;C), 310 K (36.85\u0026deg;C) and 320 K (46.85\u0026deg;C) for temperature-dependent simulations. The monochromatic light corresponding to a wavelength\u0026thinsp;~\u0026thinsp;400 nm realized by oscillating electric field[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e],\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Equa\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$E\\left(t\\right)={E}_{0}\\text{e}\\text{x}\\text{p}[-\\frac{{\\left(t-{t}_{0}\\right)}^{2}}{2{\\sigma }^{2}}]\\text{c}\\text{o}\\text{s}\\left[{\\omega }\\left(t-{t}_{0}\\right)\\right]$$\u003c/div\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ewhere ω\u0026thinsp;=\u0026thinsp;2πc/λ; σ\u0026thinsp;=\u0026thinsp;0 (for continuous wave), and \u003cem\u003eE\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e is set to be 0.2, 0.4, 0.5 and 0.6 V/nm. In addition, the analyzation of dynamic data was conducted by Gromacs tools unless otherwise specified.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e3.1. Thermal stresses\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe convergence of MD simulations was checked by temperature, pressure, density, potential energy and other thermodynamic parameters. All of these parameters indicated that the system converged within 10 ns simulations. The density and potential energy did not change dramatically at different temperature. As the temperature increasing from 290 K to 320 K, the volumes increased from 403.60 to 410.23 nm\u003csup\u003e3\u003c/sup\u003e and the densities decreased from 1024.01 to 1007.39 kg/m\u003csup\u003e3\u003c/sup\u003e. The potential energy was \u0026minus;\u0026thinsp;622591 kJ/mol, -614210 kJ/mol, -606173 kJ/mol and \u0026minus;\u0026thinsp;598067 kJ/mol for 290 K, 300 K, 310 K and 320 K, respectively.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea showed the RMSD of backbone atoms. Although the RMSD of 310 K and 320 K had more visible fluctuations than the RMSD of lower temperature, the value of RMSD remained at ~\u0026thinsp;0.15 nm. The time-dependent curves indicated that structures became relatively stable within 10 ns for all selected temperature. The calculated radius of gyration (\u003cem\u003eR\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e) was shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb for different temperatures. Generally, each \u003cem\u003eR\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e seemed time-independent with a typical constant around 1.62 nm. However, their values increased slightly with a rise in temperature, indicating structural changes might occur at high temperature. To obtain surface areas, the \u003cem\u003egmx_sasa\u003c/em\u003e command in Gromacs were performed [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e], and the time-dependent surface information for different temperature were given in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec. It could be seen that the surface area increased with the rise of temperature. At 10 ns, the surface areas were 94.68 nm\u003csup\u003e2\u003c/sup\u003e, 94.90 nm\u003csup\u003e2\u003c/sup\u003e, 95.90 nm\u003csup\u003e2\u003c/sup\u003e and 95.15 nm\u003csup\u003e2\u003c/sup\u003e for 290 K, 300 K, 310 K and 320 K, respectively.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eDictionary of protein secondary structure (DSSP) was a standard tool for the annotation of secondary structure elements from protein structures. APikL2A together with its host sHMA25 exhibited 169 residues [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. The main components of these residues were beta-sheet (~\u0026thinsp;39%), coil (~\u0026thinsp;24%), alpha-helix (~\u0026thinsp;18%). Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e showed the evolution of time-dependent secondary structure for the selected temperatures. It could be seen that the secondary structure tended to be unchanged during the MD simulations, except some unstable features observed for alpha-helix structure at 310 K.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003e3.2. Oscillating electric field stresses\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe RMSD, Rg and surface area for different oscillating electric fields at 300 K were given in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. For 0.2 V/nm and 0.4 V/nm, their time-dependent behaviors were similar to the behaviors without electric fields and could converge in 10 ns, while the time-dependent behaviors of 0.5 V/nm and 0.6 V/nm seemed not converged and presented large values. Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e showed the average of RMSD, \u003cem\u003eR\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e and surface area within 8\u0026ndash;10 ns. All of them increase with the increasing of electric field. In particular, there was a sudden increase at 0.5 V/nm, indicating the dramatically structure changes occurred. To further elaborate the changes of structure, DSSP patterns were given in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. No significantly secondary structure changes observed for 0.2 V/nm and 0.4 V/nm. However, when \u003cem\u003eE\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.5 and 0.6 V/nm, the alpha-helix structure became unstable, especially for the residues around 85 and 60. Further increasing electric field, i.e. 1 V/nm, could destroy all secondary structures, and we also found that beta-sheet and bend structure were more stable than the other structures.\u0026nbsp;\u003c/p\u003e\u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe RMSD, \u003cem\u003eR\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e and surface area for different oscillating electric fields within 8\u0026ndash;10 ns.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eField intensity (V/nm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRMSD (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1146\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1136\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1927\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.5587\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.2755\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eR\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.6042\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.6195\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.6156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.6505\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.6531\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSurface area (nm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e94.5727\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e96.5900\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e96.9242\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e104.4414\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e105.1013\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe abiotic stresses play important roles in the pathogen infection. By performing MD simulations, we exhibited the thermal and electric field (light)-dependent structural changes of APikL2A/sHMA25, a complex of rice blast effector protein with its host target from foxtail millet. All the results indicated that the structure of APikL2A/sHMA25 were relatively stable within a temperature range from 290 K to 320 K. In addition, we noticed a slightly higher surface area at higher temperature. Such an increased surface area might affect the reaction efficiency, since many protein interactions, e.g., protein-ligand binding, ion transport, and protein folding, occured at the interface between the protein and the solvent.\u003c/p\u003e\n \u003cp\u003eOn the other hand, the RMSD, Rg and surface area were significantly increased under the oscillating electric fields. This might be because of the oscillating field forced the amino acid residues to move in the direction of the field [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. Further increasing of the electric field intensity might destroy the crystal structure of protein. In APikL2A/sHMA25, this easily occurred for the residues around 85 and 60 with an alpha-helix structure, because of alpha-helixes carrying their own dipole moment, thus align themself under the oscillating electric fields. In addition, we pointed out that the beta-sheet may be the most stable in the oscillating electric fields.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by National Key R\u0026amp;D program of China (2018YFD1000705); the National Natural Science Foundation of China (32101815); HAAFS Agriculture Science and Technology Innovation Project (2022KJCXZX-GZS-1); China Agricultural Research System (CARS-06-14.5); Hebei Agricultural Research System (HBCT2018070101, HBCT2018070201) and Hebei Seed Industry Innovation Special Project(21326302D) and Hebei Province Talent Plan project (A202103001, C20210509); Talents construction project of science and technology innovation, Hebei Academy of Agriculture and Forestry Science (C22R0402).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u003c/strong\u003e Further data sets are available from the corresponding author on reasonable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability:\u0026nbsp;\u003c/strong\u003eGROMACS Software has been used under the GNU Lesser General Public License.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization: Ling Zhao, Genping Wang and Tiancong Ren; Software: Ling Zhao, Ting Zhang and Yanjie Luo; Analysis: Tiancong Ren, Lin Li. 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Innov Food Sci Emerg 35:9\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ifset.2016.03.004\u003c/span\u003e\u003cspan address=\"10.1016/j.ifset.2016.03.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-modeling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmmo","sideBox":"Learn more about [Journal of Molecular Modeling](https://www.springer.com/journal/894)","snPcode":"894","submissionUrl":"https://submission.nature.com/new-submission/894/3","title":"Journal of Molecular Modeling","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"blast effector, molecular dynamic simulations, temperature, oscillating electric field","lastPublishedDoi":"10.21203/rs.3.rs-1576885/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1576885/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e is the causal agent of rice blast. Understanding the abiotic stress influence on the resistance possess is benefit to regulate the disease. Here, effects of thermal and light (oscillating electric field) on the complex of \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e effector APikL2A/sHMA25 from foxtail millet had been investigated by molecular dynamic (MD) simulations using Gromacs package. The structure of APikL2A/sHMA25 were relatively stable within a temperature range from 290 K (16.85\u0026deg;C) to 320 K (46.85\u0026deg;C), and a slightly higher surface area was observed at higher temperature. In contrary, we found that the oscillating electric fields had a notable influence on the structure of APikL2A/sHMA25, and destructive effects could be detected at a field intensity about 0.5 V/nm. Alpha-helix structures were the least stable under oscillating electric fields, because of the existence of their dipole moment, while beta-sheet was quite be stable in the oscillating electric fields.\u003c/p\u003e","manuscriptTitle":"The temperature and light-dependent molecular dynamic investigation on foxtail millet’s blast effector APikL2A/sHMA25","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-08 16:18:42","doi":"10.21203/rs.3.rs-1576885/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions needed","date":"2022-08-22T03:39:37+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2022-07-30T15:20:10+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-07-01T09:17:28+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Journal of Molecular Modeling","date":"2022-04-21T17:46:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-04-21T16:11:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Molecular Modeling","date":"2022-04-20T08:35:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-modeling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmmo","sideBox":"Learn more about [Journal of Molecular Modeling](https://www.springer.com/journal/894)","snPcode":"894","submissionUrl":"https://submission.nature.com/new-submission/894/3","title":"Journal of Molecular Modeling","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d0ec1c74-11cd-477a-9baa-a40b62d757c8","owner":[],"postedDate":"July 8th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2022-08-22T07:40:51+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-08 16:18:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1576885","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1576885","identity":"rs-1576885","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}