Quantitative Estimation of Promoter Activity in Cannabis sativa Using Agroinfiltration-Based Transient Gene Expression.

To properly assess promoter activity, which is critical for understanding biosynthetic pathways in different plant species, we use agroinfiltration-based transient gene expression assay. We compare the activity of several known promoters in Nicotiana benthamiana with their activity in Cannabis sativa (both hemp and medicinal cannabis), which has attracted much attention in recent years for its industrial, medicinal, and recreational properties. Here we describe an optimized protocol for transient expression in Cannabis combined with a ratiometric GUS reporter system that allows more accurate evaluation of promoter activity and reduces the effects of variable infiltration efficiency.

Protein-Protein Interactions Visualized by Bimolecular Fluorescence Complementation in Arabidopsis thaliana Protoplasts from Leaf.

Bimolecular fluorescence complementation (BiFC) is a powerful tool for studying protein-protein interactions in living cells. By fusing interacting proteins to fluorescent protein fragments, BiFC allows visualization of spatial localization patterns of protein complexes. This method has been adapted to a variety of expression systems in different organisms and is widely used to study protein interactions in plant cells. The Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana (N. benthamiana) leaf cells is widely used, but in this chapter, a method for BiFC assay using Arabidopsis thaliana protoplasts is presented.

Quality evaluation of natural monomer flavors for Chinese tobacco industry based on dual mode combined Fourier transform ion cyclotron resonance mass spectrometry and isotopic fine structures.

RATIONALE: Natural monomer flavors can modify the taste of cigarettes. However, no report was published to establish the quality control method for their chemical compositions. METHODS: In this study, licorice, a traditional natural monomer flavor used in tobacco aroma processing, was selected, and the fingerprint was developed by high-performance liquid chromatography (HPLC). Next, the chemical markers of samples from different places of origin were discovered by multivariate statistical analysis. Then, its chemical constituents were identified by combination of HPLC-Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), direct infusion FT-ICR-MS (DI-FT-ICR-MS), and the technology of isotopic fine structures (IFSs). Moreover, its characteristic constituents were quantitatively analyzed using HPLC. RESULTS: The 14 common peaks were assigned in the fingerprint, and 8 of them were considered as qualitative markers by multivariate statistical analysis. A total of 42 chemical constituents were detected using HPLC-FT-ICR-MS, and 13 of them were unambiguously identified by references. Meanwhile, the elemental compositions of other eight unknown chemical components were decisively determined using IFSs. Subsequently, the contents of five characteristic constituents in 11 batches of samples were determined. CONCLUSIONS: The integration strategy established here can discover and quantify the chemical markers for improving the quality control standard of natural monomer flavor of licorice. It is expected that the strategy will be valuable for further quality control of other natural monomer flavors in Chinese tobacco industry.

Metabolic engineering of omega-3 long chain polyunsaturated fatty acids in plants using different ∆6- and ∆5-desaturases co-expressed with LPCAT from the marine diatom Phaeodactylum tricornutum.

Continuous research on obtaining an even more efficient production of very long-chain polyunsaturated fatty acids (VLC-PUFAs) in plants remains one of the main challenges of scientists working on plant lipids. Since crops are not able to produce these fatty acids due to the lack of necessary enzymes, genes encoding them must be introduced exogenously from native organisms producing VLC-PUFAs. In this study we reported, in tobacco leaves, the characterization of three distinct ∆6-desaturases from diatom Phaeodactylum tricornutum, fungi Rhizopus stolonifer and microalge Osterococcus tauri and two different ∆5-desaturases from P. tricornutum and single-celled saprotrophic eukaryotes Thraustochytrium sp. The in planta agroinfiltration of essential ∆6-desaturases, ∆6-elongases and ∆5-desaturases allowed for successful introduction of eicosapentaenoic acid (20:5∆5,8,11,14,17) biosynthesis pathway. However, despite the desired, targeted production of ω3-fatty acids we detected the presence of ω6-fatty acids, indicating and confirming previous results that all tested desaturases are not specifically restricted to neither ω3- nor ω6-pathway. Nevertheless, the additional co-expression of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) from Phaeodactylum tricornutum boosted the proportion of ω3-fatty acids in newly synthesized fatty acid pools. For the most promising genes combinations the EPA content reached at maximum 1.4% of total lipid content and 4.5% of all fatty acids accumulated in the TAG pool. Our results for the first time describe the role of LPCAT enzyme and its effectiveness in alleviating a bottleneck called 'substrate dichotomy' for improving the transgenic production of VLC-PUFAs in plants.

Exposure to pro-tobacco and anti-tobacco media messages and events and smoking behaviour among adolescents in Gambia.

BACKGROUND: Despite the widespread prevalence of adolescent smoking in Gambia, a West African country, there is limited research exploring the relationships between exposure to pro-tobacco and anti-tobacco media messages and events and smoking behaviour among young people. This study investigates the interplay of these exposures and smoking behaviour among 11-17-year-old adolescents in Gambia. METHODS: Secondary data analysis was conducted using the 2017 Gambia Global Youth and Tobacco Survey (GYTS), which included a total of 9,127 respondents. Descriptive and inferential analyses, including proportions, Pearson's chi-squared tests, and multivariable logistic regression models, were employed to estimate adjusted odds ratios (aOR) with 95% confidence intervals (CI). RESULTS: The final model revealed significant associations between exposure to anti-tobacco media messages and events and smoking behaviour. Adolescents exposed to anti-tobacco media messages had a 29% increased odds of smoking (aOR 1.29,CI = 1.08,1.53) compared to those unexposed, while exposure to anti-tobacco media events showed a 31% increased odds (aOR 1.31,CI = 1.09,1.59) compared to those unexposed. Exposure to pro-tobacco messages, such as witnessing tobacco use on TV (aOR 1.41, CI = 1.17,1.69) and owning objects with tobacco brand logos (aOR 1.49,CI = 1.19,1.86), was associated with higher odds of smoking. Covariates, including sex, age, and exposure to smoking behaviour by significant others, also demonstrated associations with smoking behaviour. Notably, male respondents showed significantly higher odds of smoking (aOR = 4.01,CI = 3.28,4.89) compared to females. Respondents aged 15 years and older had increased odds of smoking (aOR = 1.47,CI = 1.22,1.76) compared to those below 15 years old. Those whose fathers smoke displayed higher odds of smoking (aOR = 1.35, CI = 1.04,1.76) compared to individuals with non-smoking parents. Additionally, those whose closest friends smoke showed remarkably higher odds of smoking (aOR = 2.87,CI = 2.37, 3.48) compared to those without such influence. CONCLUSION: This study underscores the significant impact of exposure to both anti-tobacco and pro-tobacco media messages and events on smoking behaviour among adolescents in Gambia. However, pro-tobacco messages had a greater influence on smoking prevalence than anti-tobacco messages and events. Understanding these associations is crucial for devising effective public health interventions aimed at reducing tobacco use in this population.

Development of an attenuated potato virus Y mutant carrying multiple mutations in helper-component protease for cross-protection.

Tobacco (Nicotiana tabacum) is one of the major cash crops in China. Potato virus Y (PVY), a representative member of the genus Potyvirus, greatly reduces the quality and yield of tobacco leaves by inducing veinal necrosis. Mild strain-mediated cross-protection is an attractive method of controlling diseases caused by PVY. Currently, there is a lack of effective and stable attenuated PVY mutants. Potyviral helper component-protease (HC-Pro) is a likely target for the development of mild strains. Our previous studies showed that the residues lysine at positions 124 and 182 (K124 and K182) in HC-Pro were involved in PVY virulence, and the conserved KITC motif in HC-Pro was involved in aphid transmission. In this study, to improve the stability of PVY mild strains, K at position 50 (K50) in KITC motif, K124, and K182 were separately substituted with glutamic acid (E), leucine (L), and arginine (R), resulting in a triple-mutant PVY-HCELR. The mutant PVY-HCELR had attenuated virulence and did not induce leaf veinal necrosis symptoms in tobacco plants and could not be transmitted by Myzus persicae. Furthermore, PVY-HCELR mutant was genetically stable after six serial passages, and only caused mild mosaic symptoms in tobacco plants even at 90 days post inoculation. The tobacco plants cross-protected by PVY-HCELR mutant showed high resistance to the wild-type PVY. This study showed that PVY-HCELR mutant was a promising mild mutant for cross-protection to control PVY.

Using an ecological model of health behaviour to identify factors associated with smoking behaviour among Buddhist novices in Thailand: a cross-sectional digital survey.

OBJECTIVE: Buddhist novices reside in Buddhist temples, which are legally designated as smoke-free areas. Nevertheless, similar to other men in their age group, they are susceptible to various risk factors that lead to smoking. This digital survey aimed to examine tobacco smoking and its associated factors among Buddhist novices in Thailand. DESIGN: A cross-sectional digital survey. SETTING: 88 temple-based schools in Thailand. PARTICIPANTS: A stratified two-stage cluster sampling method was employed to select 5371 novices. Data were collected between June and August 2022 using self-administered electronic questionnaires. MEASURE: Descriptive statistics and multivariable logistic regression analysis were used to identify the associated factors. RESULTS: Overall, 32.8% of the respondents reported they had tried smoking, and the average age of initiation was 12.4 years. In the past 30 days, 25.7% had smoked any tobacco product. Multiple factors following the ecological model of health behaviour were found to be statistically associated with smoking by 37.3%. Among these were intrapersonal-level factors, such as age, living in the southern region and attempted smoking. Two were interpersonal-level factors: the smoking behaviour of close relatives, specifically parents, and their respected monks. Two were institutional-level factors: perceiving that temple-based schools are smoke-free areas and exposure to secondhand smoke. Three factors at the community and policy levels were noticed tobacco advertising at the point of sale, social media and tobacco promotion. CONCLUSION: The findings of this study support the development of comprehensive intervention programmes that address the multiple factors to prevent Buddhist novices from smoking.

Overexpression of the WRKY transcription factor gene NtWRKY65 enhances salt tolerance in tobacco (Nicotiana tabacum).

BACKGROUND: Salt stress severely inhibits plant growth, and the WRKY family transcription factors play important roles in salt stress resistance. In this study, we aimed to characterize the role of tobacco (Nicotiana tabacum) NtWRKY65 transcription factor gene in salinity tolerance. RESULTS: This study characterized the role of tobacco (Nicotiana tabacum) NtWRKY65 transcription factor gene in salinity tolerance using four NtWRKY65 overexpression lines. NtWRKY65 is localized to the nucleus, has transactivation activity, and is upregulated by NaCl treatment. Salinity treatment resulted in the overexpressing transgenic tobacco lines generating significantly longer roots, with larger leaf area, higher fresh weight, and greater chlorophyll content than those of wild type (WT) plants. Moreover, the overexpressing lines showed elevated antioxidant enzyme activity, reduced malondialdehyde content, and leaf electrolyte leakage. In addition, the Na+ content significantly decreased, and the K+/Na+ ratio was increased in the NtWRKY65 overexpression lines compared to those in the WT. These results suggest that NtWRKY65 overexpression enhances salinity tolerance in transgenic plants. RNA-Seq analysis of the NtWRKY65 overexpressing and WT plants revealed that NtWRKY65 might regulate the expression of genes involved in the salt stress response, including cell wall component metabolism, osmotic stress response, cellular oxidant detoxification, protein phosphorylation, and the auxin signaling pathway. These results were consistent with the morphological and physiological data. These findings indicate that NtWRKY65 overexpression confers enhanced salinity tolerance. CONCLUSIONS: Our results indicated that NtWRKY65 is a critical regulator of salinity tolerance in tobacco plants.

Rice yellow mottle virus is a suitable amplicon vector for an efficient production of an anti-leishmianiasis vaccine in Nicotiana benthamiana leaves.

BACKGROUND: Since the 2000's, plants have been used as bioreactors for the transient production of molecules of interest such as vaccines. To improve protein yield, "amplicon" vectors based on plant viruses are used. These viral constructs, engineered to carry the gene of interest replicate strongly once introduced into the plant cell, allowing significant accumulation of the protein. Here, we evaluated the suitability of the monocot-infecting RNA virus Rice yellow mottle virus (RYMV) as an amplicon vector. The promastigote surface antigen (PSA) of the protozoan Leishmania was considered as a protein of interest due to its vaccine properties against canine leishmaniasis. RESULTS: Since P1 (ORF1) and CP (ORF3) proteins are not strictly necessary for viral replication, ORF1 was deleted and the PSA gene was substituted to ORF3 in the RYMV-based vector. We evaluated its expression in the best described plant bioreactor system, Nicotiana benthamiana which, unlike rice, allows transient transformation by Agrobacterium. Despite not being its natural host, we demonstrated a low level of RYMV-based vector replication in N. benthamiana leaves. Under optimized ratio, we showed that the P19 silencing suppressor in combination with the missing viral CP ORF significantly enhanced RYMV amplicon replication in N. benthamiana. Under these optimized CP/P19 conditions, we showed that the RYMV amplicon replicated autonomously in the infiltrated N. benthamiana cells, but was unable to move out of the infiltrated zones. Finally, we showed that when the RYMV amplicon was expressed under the optimized conditions we set up, it allowed enhanced PSA protein accumulation in N. benthamiana compared to the PSA coding sequence driven by the 35S promoter without amplicon background. CONCLUSION: This work demonstrates that a non-dicot-infecting virus can be used as an amplicon vector for the efficient production of proteins of interest such as PSA in N. benthamiana leaves.

Genome-wide characterization and expression analysis of the HD-Zip II gene family in response to drought and GA3 stresses in Nicotiana tabacum.

BACKGROUND: Homeodomain-leucine ZIPper (HD-ZIP) transcription factors play crucial roles in plant growth, development, and stress responses. The HD-ZIP family is categorised into four groups (HD-ZIP I-IV). While extensive genome-wide studies have been conducted on the HD-ZIP I, III, and IV subfamily in Nicotiana tabacum (tobacco), comprehensive reports on the HD-ZIP II subfamily genes are limited. METHODS: Bioinformatics resources and tools were utilised to analyse molecular characteristics, phylogenetic homology, and protein interactions. Expression pattern analyses in various tissues and the relative expression of NtHD-ZIP II genes under drought and GA3 treatment were assessed by qRT-PCR. RESULTS: In this study, 24 HD-ZIP II members were systematically identified and categorised into seven independent clades through phylogenetic analysis involving tobacco and other plant species. We found that 19 NtHD-ZIP II genes exhibited tissue-specific expression. The transcripts of NtHD-ZIPII3, 4, 14, 23, 24 were notably induced under the drought treatments, while those of NtHD-ZIPII7, 11, 12, 20 were suppressed. Furthermore, NtHD-ZIPII15 transcripts decreased following GA3 treatment, whereas the transcripts of NtHD-ZIPII7, 8, 11, 12 were induced after GA3 treatment. Notably, an increase in trichomes was observed in tobacco leaves treated with GA3 and subjected to drought. CONCLUSIONS: The expression levels of some HD-ZIP II genes were altered, and an increase in glandular trichomes was induced under GA3 and drought treatments in tobacco. Overall, our findings provide insights into the expression patterns of NtHD-ZIP II genes and will facilitate their functional characterisation in future studies.

Comparative evaluation of ten blood biomarkers of inflammation in regular heated tobacco users and non-smoking healthy males-a pilot study.

Heated tobacco products (HTPs) are novel tobacco products that are alternatives to cigarettes. The study aimed to investigate the effect of HTPs on blood biomarkers of inflammation as well as to provide a comparative evaluation between daily heated tobacco users and healthy men who do not use nicotine products. This case-control study was carried out among 92 healthy males in Poland (Lodz-Province) aged 20-56 years: 44 daily heated tobacco users (daily use in the past 90 days) and 48 controls who do not use nicotine products. The history of use of the nicotine-containing products was self-reported and verified using a saliva cotinine test. A 20 ml blood sample was collected and the levels of ten blood biomarkers were analyzed. Among all heated tobacco users (n = 44), only the levels of interleukin 8 (IL-8) were significantly higher when compared to controls: 6.86 vs. 3.95 (p = 0.01). Among exclusive heated tobacco users (n = 33), the levels of IL-8 were also significantly higher when compared to controls: 7.76 vs. 3.95 (p = 0.01). IL-8 level was positively correlated (r = 0.37; p = 0.01) with the daily number of heated tobacco sticks. Out of 10 different biomarkers of inflammation, only IL-8 levels were significantly elevated in heated tobacco use compared to controls.

Creating and Implementing a Community-Focused, Culturally Tailored Health Marketing Campaign to Address Menthol Cigarette Use in Los Angeles County.

Introduction: Menthol tobacco products have been marketed disproportionately to communities of color for decades. Methods: In Los Angeles County, California, a health marketing campaign, which used glossy visuals and attractive people in appealing poses, reminiscent of tobacco marketing tactics, was created and implemented to educate smokers on the health risks of using menthol cigarettes. The campaign encouraged smokers to make a quit attempt by offering access to free or low-cost resources through the Kick It California quitline and the LAQuits website (laquits.com). A survey tailored for public health professionals and community members from the approximately 382,000 people in the county who smoked menthol cigarettes and were exposed to their smoke (our primary audience) was administered to generate insights about this problem. Survey data were used to finesse the campaign creative materials prior to launch. Advertisement exposures, website visits, and quitline call volume were monitored and tabulated to assess the performance of the campaign. Results: At the conclusion of its initial run (February-April 2021), the "Done with Menthol" campaign had garnered more than 66 million impressions, received approximately 56,000 clicks on its various digital media platforms, and had click-through rates that surpassed industry benchmarks. The quitline call volume for African American and Latino subgroups were 1.9 and 1.8 times higher than the average inbound call volume for corresponding months during 2018 and 2019, respectively. In its second run (May-June 2023), the campaign garnered approximately 11 million additional impressions. Conclusions: Despite having a lower budget and fewer resources than the tobacco industry, the "Done with Menthol" campaign attained excellent reach and offered free, low-cost, and accessible resources to county residents interested in tobacco use cessation.

Our perception may not be reality: A longitudinal study of the relationship between perceived and actual change in smoking behavior.

INTRODUCTION: Results of the impact of lockdowns and stay-at-home orders during the COVID-19 pandemic on changes in cigarette smoking are mixed. Previous studies examining smoking changes during the early stages of the pandemic in 2020 have mainly focused on smoker's perception of changes in cigarette consumption. Such measure has not been widely used in other contexts, and therefore we aim to compare the discrepancy between smokers' perceived changes in cigarette smoking and the actual change in the number of cigarettes smoked, using repeated measurements. METHODS: We included 134 smokers from the French TEMPO cohort with repeated measurements of their perceived changes in smoking habits during the first phase of the COVID-19 pandemic and the number of cigarettes smoked repeatedly from March to May 2020. We used generalized estimation equations (GEE) to examine the association between changes in the number of cigarettes smoked and the odds of mismatched answers. RESULTS: The results suggest that at each study wave, 27-45% of participants provided mismatching answers between their perceived change in smoking habits and the actual change in the number of cigarettes smoked daily, measured repeatedly. Results from GEE analysis demonstrated that a mismatching assessment of smoking behavior was elevated among those who had an increase (OR = 2.52 [1.37;4.65]) or a decrease (OR = 5.73 [3.27;10.03]) in number of cigarettes smoked. DISCUSSION: Our findings highlight the possibility of obtaining different results depending on how changes in tobacco smoking are measured. This highlights the risk of underestimating the actual changes in cigarette smoking during the COVID-19 pandemic, but also more generally when validating public health interventions or smoking cessation programs. Therefore, objective measures such as the actual consumption of psychoactive substances should be utilized, preferably on a longitudinal basis, to mitigate recall bias.

Countering the influence of tobacco.

Tobacco advertising regulations need to adapt if they are to meet the challenges posed by a rapidly evolving social media and tobacco product landscape. Gary Humphreys reports.

AnDREB5.1, a A5 group DREB gene from desert shrub Ammopiptanthus nanus, confers osmotic and cold stress tolerances in transgenic tobacco.

The Dehydration-Responsive Element Binding (DREB) subfamily of transcription factors plays crucial roles in plant abiotic stress response. Ammopiptanthus nanus (A. nanus) is an eremophyte exhibiting remarkable tolerance to environmental stress and DREB proteins may contribute to its tolerance to water deficit and low-temperature stress. In the present study, an A. nanus DREB A5 group transcription factor gene, AnDREB5.1, was isolated and characterized in terms of structure and function in abiotic stress tolerance. AnDREB5.1 protein is distributed in the nucleus, possesses transactivation capacity, and is capable of binding to DRE core cis-acting element. The transcription of AnDREB5.1 was induced under osmotic and cold stress. Tobacco seedlings overexpressing AnDREB5.1 displayed higher tolerance to cold stress, osmotic stress, and oxidative stress compared to wild-type tobacco (WT). Under osmotic and cold stress, overexpression of AnDREB5.1 increased antioxidant enzyme activity in tobacco leaves, inhibiting excessive elevation of ROS levels. Transcriptome sequencing analysis showed that overexpression of AnDREB5.1 raised the tolerance of transgenic tobacco seedlings to abiotic stress by regulating multiple genes, including antioxidant enzymes, transcription factors, and stress-tolerant related functional genes like NtCOR413 and NtLEA14. This study provides new evidence for understanding the potential roles of the DREB A5 subgroup members in plants.

National prevalence of smoking among adolescents at tobacco tax increase and COVID-19 pandemic in South Korea, 2005-2022.

Prior research has predominantly focused on the overall effects of the tobacco tax increase and the COVID-19 pandemic on adolescent smoking behavior. However, there is a need to examine both the immediate and sustained associations of these two factors on subgroups of adolescents, employing an interrupted time-series model. We aimed to investigate the immediate and sustained association of tobacco tax increase and the COVID-19 pandemic on adolescent smoking prevalence. This study utilized data from the Korea Youth Risk Behavior Web-Based Survey to analyze the prevalence of current smoking among all participants (CSP) and the prevalence of daily smoking among current smokers (DSP) of Korean adolescents (n = 1,159,995; mean, age 14.99; male 51.5%) over 18 years from 2005 to 2022. The study examined 18-year trends in CSP and DSP among Korean adolescents, emphasizing the influences of the 2015 tobacco tax increase and the COVID-19 pandemic, using ß coefficients and their differences (ßdiff) from an interrupted time-series ARIMA model. While CSP exhibited a decreasing trend, DSP exhibited an increasing trend. Tobacco tax increase was associated with both the short and long terms in smoking prevalence, however, the short-term association on prevalence (CSP, - 3.076 [95% CI, - 3.707 to - 2.445]; DSP, - 4.112 [95% CI, - 6.488 to - 1.735]) was stronger. The pandemic was associated with an immediate increase in DSP (9.345 [95% CI, 5.285-13.406]). These effects were strongest among adolescents from low economic status and those exposed to familial secondhand smoking. Supportive programs for adolescents in low-income families will help overcome the effects associated with the pandemic. As a tobacco tax increase was associated with a reduction in smoking prevalence, this could be one method to overcome the effects of the pandemic.

Occupational exposures among women beedi workers in Mysore District, India: A mixed-methods study protocol.

Beedi is the most common smoking form of tobacco used in India. The rolling of beedis is performed primarily by women in settings that lack occupational safeguards. The aims of this protocol are to establish methods for the study of occupational exposures among women beedi workers and their experiences and challenges working with unburnt tobacco. This protocol employs a convergent parallel mixed-methods approach. Qualitatively, we plan to explore the experiences and challenges faced by women beedi workers using photovoice, a community based participatory method. Occupational exposures to pesticides will be assessed through the use of silicone wristbands worn for seven days by workers, and exposure to toxic metals and metalloids will be assessed in dust samples collected in the homes of workers. The outcomes will be analyzed to form policy recommendations to improve the occupational health of women beedi workers.

Three new discovery effector proteins from Candidatus Liberibacter asiaticus psy62 inhibit plant defense through interaction with AtCAT3 and AtGAPA.

KEY MESSAGE: We identify three SDEs that inhibiting host defence from Candidatus Liberibacter asiaticus psy62, which is an important supplement to the pathogenesis of HLB. Candidatus Liberibacter asiaticus (CLas) is the main pathogen of citrus Huanglongbing (HLB). 38 new possible sec-dependent effectors (SDEs) of CLas psy62 were predicted by updated predictor SignalP 5.0, which 12 new SDEs were found using alkaline phosphate assay. Among them, SDE4310, SDE4435 and SDE4955 inhibited hypersensitivity reactions (HR) in Arabidopsis thaliana (Arabidopsis, At) and Nicotiana benthamiana leaves induced by pathogens, which lead to a decrease in cell death and reactive oxygen species (ROS) accumulation. And the expression levels of SDE4310, SDE4435, and SDE4955 genes elevated significantly in mild symptom citrus leaves. When SDE4310, SDE4435 and SDE4955 were overexpressed in Arabidopsis, HR pathway key genes pathogenesis-related 2 (PR2), PR5, nonexpressor of pathogenesis-related 1 (NPR1) and isochorismate synthase 1 (ICS1) expression significantly decreased and the growth of pathogen was greatly increased relative to control with Pst DC3000/AvrRps4 treatment. Our findings also indicated that SDE4310, SDE4435 and SDE4955 interacted with AtCAT3 (catalase 3) and AtGAPA (glyceraldehyde-3-phosphate dehydrogenase A). In conclusion, our results suggest that SDE4310, SDE4435 and SDE4955 are CLas psy62 effector proteins that may have redundant functions. They inhibit ROS burst and cell death by interacting with AtCAT3 and AtGAPA to negatively regulate host defense.

Chinese cherry CpMYB44-CpSPDS2 module regulates spermidine content and florescence in tobacco.

The flower bud differentiation plays a crucial role in cherry yield and quality. In a preliminary study, we revealed the promotion of spermidine (Spd) in bud differentiation and quality. However, the molecular mechanism underlying Spd regulating cherry bud differentiation remains unclear. To address this research gap, we cloned CpSPDS2, a gene that encodes Spd synthase and is highly expressed in whole flowers and pistils of the Chinese cherry (cv. 'Manaohong'). Furthermore, an overexpression vector with this gene was constructed to transform tobacco plants. The findings demonstrated that transgenic lines exhibited higher Spd content, an earlier flowering time by 6 d, and more lateral buds and flowers than wild-type lines. Additionally, yeast one-hybrid assays and two-luciferase experiments confirmed that the R2R3-MYB transcription factor (CpMYB44) directly binds to and activates the CpSPDS2 promoter transcription. It is indicated that CpMYB44 promotes Spd accumulation via regulating CpSPDS2 expression, thus accelerating the flower growth. This research provides a basis for resolving the molecular mechanism of CpSPDS2 involved in cherry bud differentiation.