Resistant Transgenic Plants Research Articles

Harnessing environmental‐RNAi to win an enduring battle: Plant versus pathogen

AbstractPlant‐microbiome interactions are sophisticated phenomena with unrevealed features. Current progress in RNA biology has enlightened the impact of exchanging small RNAs between plants and their associated microbes in altering the battlefield conditions in their favour. The discovery of RNAi and the release of resistant transgenic plants have raised public health concerns rushing to develop less‐invasive control strategies. As an alternative, the topical application of small RNAs to inhibit gene expression via non‐transformative RNAi in pathogens and pests, opens new horizons in plant disease management. The present review highlights how plants and their microbial associates send and receive small RNA molecules and that sRNAs and delivery techniques characteristics provide an effective disease control.

Development of marker-free insect resistant transgenic okra (Abelmoschus esculentus L. Moench) expressing the cry1Ac gene and identification of vector backbone-free events.

Agrobacterium-mediated co-transformation method was used to generate marker-free insect resistant transgenic okraplants expressing the cry1Ac gene. The cry1Ac gene was borne on the T-DNA of one plasmid while nptII and uidA (GUS) marker genes were present on the T-DNA of a second plasmid. Putative transgenic plants were screened by histochemical GUS assay for expression of -glucuronidase and 32 transgenic events were positive for GUS in which 21 transgenic events were positive in ELISA for the presence of Cry1Ac protein. Out of 21 Cry1Ac positive T0 events, three events displayed Mendelian inheritance of the transgenes in (9:3:3:1 ratio) T1 generation for Cry1Ac and GUS. Selected events were chosen for further genetic and molecular analysis. The cry1Ac and marker genes were found to segregate independently, of each other in 10 events in T1 generation out of 11 Cry1Ac gene inheriting events analysed indicating that the two T-DNAs insertions were genetically unlinked and identification of marker-free plants were possible in these 10 events. The marker-free nature and vector backbone-free Bt events (clean T-DNA insertions carrying cry1Ac gene) were confirmed by Southern analysis using suitable probes. The plants from selected transgenic events were rigorously screened in whole plant insect bioassays using the larvae of shoot and fruit borer, Earias vittella, an important pest of okra. Insect bioassays indicated 100% larval mortality without any infestation in five of the transgenic events and two events showed 5 to 10 percent infestation establishing the insect resistant nature of the transgenic plants. Finally the events inheriting transgenes in Mendelian fashion were characterized further and marker-free and vector backbone-free events were identified showing complete protection from the target pest Earias vittella in whole-plant insect bioassays. Quantification of Cry1Ac protein levels in the plant parts of selected events (lines) wasconsistent with the results of bioassays. Further, two lines identified in this study met the criteria for inclusion in commercial breeding programs.

The miR169n-NF-YA8 regulation module involved in drought resistance in Brassica napus L

As an ancient and conserved plant microRNA (miRNA) family, miR169 targets nuclear factor Y subunit alpha (NF-YA) family members. The miR169-NF-YA module is associated with plant development and various stress responses. However, the function of miR169 in response to drought stress in rapeseed (Brassica napus L.) is unclear. In the present study, we showed that miR169n acted as a negative regulator of drought resistance in rapeseed by targeting a nuclear factor Y-A gene, NF-YA8. miR169n was strongly down-regulated by drought stress. Expression of a miR169n target mimicry construct (MIM169n) which functioned as a sponge to trap miR169n resulted in enhanced resistance of transgenic plants to both osmotic stress at the post-germination stage and drought stress at the seedling stage. MIM169n plants had a higher relative water content (RWC) and proline content, lower relative electrolyte leakage (REL), and showed higher antioxidative capability compared with those of control (CK) plants under drought stress. Moreover, NF-YA8 was verified as a target of miR169n, and overexpression of NF-YA8 led to improved tolerance of rapeseed to osmotic stress at the post-germination stage. Overall, our findings implied that the miR169n-NF-YA8 regulatory module could serve as a potential target for genetic improvement of drought resistance in B. napus.

Kanamycin application features for the selective screening of genetically modified Populus × berolinensis

The study describes the features of the kanamycin application for the effective selection of transgenic poplar expressing nptII gene. It has been shown that kanamycin concentration of 50 mg L−1 in the nutrient medium is the most suitable concentration for the effective selection of transgenic Berlin poplar plants for both regeneration and rooting stages. At the same time, 50 mg L−1 of kanamycin in the nutrient medium may result in the loss of the less resistant transgenic plants, so lower concentrations may be also applied. Kanamycin concentrations of 25 and 35 mg L−1 can give a false positive signal of transgenesis. Transgenic poplars expressing nptII gene can be effectively regenerated on nutrient media with kanamycin at concentrations up to 100 mg L−1.

Transcriptome and DNA Methylome Reveal Insights Into Phytoplasma Infection Responses in Mulberry (Morus multicaulis Perr.).

To reveal whether the response of mulberry to phytoplasma infection is associated with genome-wide DNA methylation changes, the methylome and transcriptome patterns of mulberry in response to phytoplasma infection were explored. Though the average methylation level of the infected leaves showed no significant difference from that of healthy leaves, there were 1,253 differentially methylated genes (DMGs) and 1,168 differentially expressed genes (DEGs) in the infected leaves, and 51 genes were found simultaneously to be differently methylated and expressed. It was found that the expression of G-type lectin S-receptor-like serine/threonine protein kinase gene (Mu-GsSRK) was increased, but its methylation level was decreased in the pathogen-infected or salicylic acid (SA)-treated leaves. Overexpression of Mu-GsSRK in Arabidopsis and in the hairy roots of mulberry enhanced transgenic plant resistance to the phytoplasma. Moreover, overexpression of Mu-GsSRK enhanced the expressions of pathogenesis-related protein 1, plant defensin, and cytochrome P450 protein CYP82C2 genes in transgenic plants inoculated with pathogens, which may contribute to the enhanced disease resistance against various pathogens. Finally, the DNA methylation dynamic patterns and functions of the differentially expressed and methylated genes were discussed. The results suggested that DNA methylation has important roles in mulberry responses to phytoplasma infection.

GhKWL1 Upregulates GhERF105 but Its Function Is Impaired by Binding with VdISC1, a Pathogenic Effector of Verticillium dahliae.

Verticillium wilt, caused by Verticillium dahliae, is a devastating disease for many important crops, including cotton. Kiwellins (KWLs), a group of cysteine-rich proteins synthesized in many plants, have been shown to be involved in response to various phytopathogens. To evaluate genes for their function in resistance to Verticillium wilt, we investigated KWL homologs in cotton. Thirty-five KWL genes (GhKWLs) were identified from the genome of upland cotton (Gossypium hirsutum). Among them, GhKWL1 was shown to be localized in nucleus and cytosol, and its gene expression is induced by the infection of V. dahliae. We revealed that GhKWL1 was a positive regulator of GhERF105. Silencing of GhKWL1 resulted in a decrease, whereas overexpression led to an increase in resistance of transgenic plants to Verticillium wilt. Interestingly, through binding to GhKWL1, the pathogenic effector protein VdISC1 produced by V. dahliae could impair the defense response mediated by GhKWL1. Therefore, our study suggests there is a GhKWL1-mediated defense response in cotton, which can be hijacked by V. dahliae through the interaction of VdISC1 with GhKWL1.

English

One of the most crucial threats limiting the sustainable production of cotton is cotton leaf curl disease (CLCuD). There is dire need to produce a resistant variety that can combat CLCuD. For this purpose, virus resistant transgenic cotton plants (MNH-786) with C4 gene construct at T3 generation were selected and sown. Young fresh leaves of multigenic variety of MNH-786 were collected to confirm the transformed construct. Infected whiteflies were used for spreading on transgenic cotton MNH-786 variety with C4 construct to check percentage of infection. Whiteflies were collected from infected cotton plants showing CLCuD and reared in lab to increase the population of whiteflies. After 15 days of feeding, infected leaves of transgenic plants were collected and total DNA of infected leaves of transgenic cotton plant with virus load was extracted. At maturity, data of morphological characteristic was taken from the transgenic cotton plants of MNH-786 and control plants. Resistant transgenic cotton plants showed < 0.5% disease index and recorded more plant height in field condition. Total number of bolls per plant was 20% more in tolerant plants and 40% more in resistant plants as compared to susceptible plants. Molecular analysis of transgenic plants showed clear evidence that expression of construct 4 virus resistant gene against begomoviruses in resistant and tolerant group of transgenic plants was more as compared to susceptible group and control. © 2021 Friends Science Publishers

Pyramiding the antimicrobial PR1aCB and AATCB genes in 'Tarocco' blood orange (Citrus sinensis Osbeck) to enhance citrus canker resistance.

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a major bacterial disease responsible for substantial economic losses in citrus-producing areas. To breed transgenic citrus plants with enhanced resistance to citrus canker, two antimicrobial peptide genes, PR1aCB and AATCB, were incorporated into 'Tarocco' blood orange (Citrus sinensis Osbeck) plants via co-transformation and sequential re-transformation. The presence of PR1aCB and AATCB in double transgenic plants was confirmed by PCR. The expression of PR1aCB and AATCB in double transformants was demonstrated by quantitative real-time PCR. An in vivo disease resistance assay involving the injection of Xcc revealed that the double transformants were more resistant to citrus canker than the single gene transformants and wild-type plants. An analysis of the bacterial population indicated that the enhanced citrus canker resistance of the double transformants was due to inhibited Xcc growth. These results proved that the pyramiding of multiple genes is a more effective strategy for increasing the disease resistance of transgenic citrus plants than single gene transformations.

Endophytic bacteria associated with the enhanced cadmium resistance in NHX1- overexpressing tobacco plants

Human industrial activities have left large area of land polluted with cadmium (Cd), which poses tremendous threat to ecosystem. Na+/H+ antiporter 1 (NHX1) was a transporter located on vacuoles, which mediated the compartmentation of Na+ into vacuole and transportation of H+ into the cytoplasm. And the overexpressing of its code gene (NHX1 gene) was previously identified to enhance Cd resistance in tobacco (Nicotiana tabacum) plants possibly by maintaining the change of ion content. However, the mechanisms of Cd resistance in NHX1-overexpressing plants have not been well elucidated yet. In this study, the endophytic bacteria communities, the activities of soil enzymes and the antioxidant enzymes were characterized to explore possible mechanisms of the enhanced Cd resistance in NHX1-overeexpressing tobacco plants. Some differences in the endophytic bacterial community structure in roots of transgenic and WT tobaccos under Cd stress were observed, in which, Sphingomonas, Steroidobacter and Actinoplanes were proposed as vital endophytic bacteria for enhancing Cd resistance of transgenic tobacco plants. In addition, the soil enzymes were increased in the rhizospheric soil around the transgenic tobacco plants compared to WT plants under Cd stress. Under Cd exposure, the SOD and CAT activities were 1.65 times and 1.88 times of that in WT plants, respectively. The content of MDA and H2O2 were also observed to be 35 % and 28 % lower in transgenic tobacco roots compared to WT plants under Cd stress. Moreover, the Cd content of transgenic plants decreased by 38 % under Cd stress compared to WT plants. These findings suggested that the endophytic bacteria communities associated with the NHX1-overexpressing plants might lead to the increased Cd resistance by enhancing the rhizosphere soil enzyme activities, strengthening the antioxidative defense system, and better maintaining of the ion changes. Taken together, this study suggested that the enhanced Cd resistance was observed in transgenic tobacco plants associated with the increase of soil enzyme activities and changes of endophytic bacteria community, and the possible mechanisms involved in this process were also discussed.

Analysis of drought and salt-alkali tolerance in tobacco by overexpressing WRKY39 gene from Populus trichocarpa

ABSTRACT WRKY is one of the largest families of transcription factors in plants. It not only regulates plant growth and development but also participates in the regulation of plant defense against biological and abiotic stresses. In this study, research was aimed to overexpress WRKY39 gene of P. trichocarpa (PtWRKY39) and to identify its important role played in drought and saline-alkali tolerance in tobacco model plant. Under the control of CaMV35S promoter, the overexpression of PtWRKY39 gene was increased to more than 10 times in T3 generation of transgenic tobacco plant. The drought resistance and saline-alkali tolerance were evidenced in overexpressed PtWRKY39 transgenic lines at germination/seedling stage. The overall germination rate, fresh weight, and chlorophyll contents of overexpressed lines were significantly higher while the level of malondialdehyde was significantly lower in PtWRKY39 transgenic lines than that of wild type (WT) lines. The content of H2O2 in leaves was detected by the 3, 3-Diaminobenzidine method showed that the overexpression of PtWRKY39 gene could reduce the accumulation of ROS (mainly H2O2) and enhance salt-alkali tolerance. Phenotypic analysis at 7-leaf pot transgenic seedlings stage treated with the saline-alkali soil extract and salt NaCl under root irrigation stress, revealed growth of the transgenic line was significantly higher than that of WT. This work concludes that overexpression of PtWRKY39 gene can improve the regulation of drought resistance and saline-alkali tolerance of transgenic plants during seed germination and vegetative growth.

BiP-overexpressing soybean plants display accelerated hypersensitivity response (HR) affecting the SA-dependent sphingolipid and flavonoid pathways

Biotic and abiotic environmental stresses have limited the increase in soybean productivity. Overexpression of the molecular chaperone BiP in transgenic plants has been associated with the response to osmotic stress and drought tolerance by maintaining cellular homeostasis and delaying hypersensitive cell death. Here, we evaluated the metabolic changes in response to the hypersensitivity response (HR) caused by the non-compatible bacteria Pseudomonas syringae pv. tomato in BiP-overexpressing plants. The HR-modified metabolic profiles in BiP-overexpressing plants were significantly distinct from the wild-type untransformed. The transgenic plants displayed a lower abundance of HR-responsive metabolites as amino acids, sugars, carboxylic acids and signal molecules, including p-aminobenzoic acid (PABA) and dihydrosphingosine (DHS), when compared to infected wild-type plants. In contrast, salicylic acid (SA) biosynthetic and signaling pathways were more stimulated in transgenic plants, and both pathogenesis-related genes (PRs) and transcriptional factors controlling the SA pathway were more induced in the BiP-overexpressing lines. Furthermore, the long-chain bases (LCBs) and ceramide biosynthetic pathways showed alterations in gene expression and metabolite abundance. Thus, as a protective pathway against pathogens, HR regulation by sphingolipids and SA may account at least in part by the enhanced resistance of transgenic plants. GmNAC32 transcriptional factor was more induced in the transgenic plants and it has also been reported to regulate flavonoid synthesis in response to SA. In fact, the BiP-overexpressing plants showed an increase in flavonoids, mainly prenylated isoflavones, as precursors for phytoalexins. Our results indicate that the BiP-mediated acceleration in the hypersensitive response may be a target for metabolic engineering of plant resistance against pathogens.

MaMAPK3-MaICE1-MaPOD P7 pathway, a positive regulator of cold tolerance in banana

BackgroundBanana is a tropical fruit with a high economic impact worldwide. Cold stress greatly affects the development and production of banana.ResultsIn the present study, we investigated the functions of MaMAPK3 and MaICE1 involved in cold tolerance of banana. The effect of RNAi of MaMAPK3 on Dajiao (Musa spp. ‘Dajiao’; ABB Group) cold tolerance was evaluated. The leaves of the MaMAPK3 RNAi transgenic plants showed wilting and severe necrotic symptoms, while the wide-type (WT) plants remained normal after cold exposure. RNAi of MaMAPK3 significantly changed the expressions of the cold-responsive genes, and the oxidoreductase activity was significantly changed in WT plants, while no changes in transgenic plants were observed. MaICE1 interacted with MaMAPK3, and the expression level of MaICE1 was significantly decreased in MaMAPK3 RNAi transgenic plants. Over-expression of MaICE1 in Cavendish banana (Musa spp. AAA group) indicated that the cold resistance of transgenic plants was superior to that of the WT plants. The POD P7 gene was significantly up-regulated in MaICE1-overexpressing transgenic plants compared with WT plants, and the POD P7 was proved to interact with MaICE1.ConclusionsTaken together, our work provided new and solid evidence that MaMAPK3-MaICE1-MaPOD P7 pathway positively improved the cold tolerance in monocotyledon banana, shedding light on molecular breeding for the cold-tolerant banana or other agricultural species.

Overexpressing a NPR1-like gene from Citrus paradisi enhanced Huanglongbing resistance in C. sinensis.

Overexpression of CiNPR4 enhanced resistance of transgenic citrus plants to Huanglongbing by perceiving the salicylic acid and jasmonic acid signals and up-regulating the transcriptional activities of plant-pathogen interaction genes. Developing transgenic citrus plants with enhanced immunity is an efficient strategy to control citrus Huanglongbing (HLB). Here, a nonexpressor of pathogenesis-related gene 1 (NPR1) like gene from HLB-tolerant 'Jackson' grapefruit (Citrus paradisi Macf.), CiNPR4, was introduced into 'Wanjincheng' orange (Citrus sinensis Obseck). CiNPR4 expression was determined in transgenic citrus plants using quantitative real-time PCR analyses. The Candidatus Liberibacter asiaticus (CLas) pathogen of HLB was successfully transmitted to transgenic citrus plants by grafting infected buds. HLB symptoms developed in transgenic and wild-type (WT) plants by 9months after inoculation. A CLas population analysis showed that 26.9% of transgenic lines exhibited significantly lower CLas titer levels compared with the CLas-infected WT plants at 21months after inoculation. Lower starch contents and anatomical aberration levels in the phloem were observed in transgenic lines having enhanced resistance compared with CLas-infected WT plants. CiNPR4 overexpression changed the jasmonic acid, but not salicylic acid, level. Additionally, the jasmonic acid and salicylic acid levels increased after CLas infection. Transcriptome analyses revealed that the enhanced resistance of transgenic plants to HLB resulted from the up-regulated transcriptional activities of plant-pathogen interaction-related genes.

Over expression of a Chitinase 2 gene from Chinese Wild Strawberry improves resistance to anthracnose disease in transgenic Arabidopsis thaliana

Strawberry (Fragaria × ananassa Duch.) is one of the most widely cultivated fruit crop. Anthracnose caused by Colletotrichum spp. is a devastating disease of strawberry, causing large-scale strawberry losses worldwide. Chitinases act as defence proteins and are crucial for plant response to pathogens. Here, we isolated a class V Chitinase gene (designed as FnCHIT2, GenBank accession number MN709779) from Chinese wild diploid strawberry Fragaria nilgerrensis Schlecht (F. nilgerrensis), a species that exhibits high tolerance to anthracnose. Gene expression analysis showed that FnCHIT2 expression was highly induced after Colletotrichum gloeosporiodes inoculation and salicylic acid (SA) treatment. Subcellular localization analysis revealed the presence of FnCHIT2 in the plasma membrane. Recombinant FnCHIT2 protein was successfully expressed in E. coli Rosetta (DE3). Furthermore, we transformed FnCHIT2 into Col-0 wild type A. thaliana to perform functional analysis and evaluated the functions of Colletotrichum higginsianum and Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). FnCHIT2 overexpression in A. thaliana showed enhanced resistance to C. higginsianum and Pst DC3000. Enhanced disease resistance of FnCHIT2 transgenic plants to C. higginsianum was correlated with pathogenesis-related gene 1 (AtPR1) and plant defensin 1.2 (AtPDF1.2) gene expression levels. These results provide evidence that FnCHIT2 may play an important role in response to fungal pathogens in strawberry. Our study provides an important theoretical reference for future strawberry resistance breeding.

Characterization of the DREBA4-Type Transcription Factor (SlDREBA4), Which Contributes to Heat Tolerance in Tomatoes.

Dehydration-responsive element binding (DREB) transcription factors play crucial regulatory roles in abiotic stress. The only DREB transcription factor in tomato (Solanum lycopersicum), SlDREBA4 (Accession No. MN197531), which was determined to be a DREBA4 subfamily member, was isolated from cv. Microtom using high-temperature-induced digital gene expression (DGE) profiling technology. The constitutive expression of SlDREBA4 was detected in different tissues of Microtom plants. In addition to responding to high temperature, SlDREBA4 was up-regulated after exposure to abscisic acid (ABA), cold, drought and high-salt conditions. Transgenic overexpression and silencing systems revealed that SlDREBA4 could alter the resistance of transgenic Microtom plants to heat stress by altering the content of osmolytes and stress hormones, and the activities of antioxidant enzymes at the physiologic level. Moreover, SlDREBA4 regulated the downstream gene expression of many heat shock proteins (Hsp), as well as calcium-binding protein enriched in the pathways of protein processing in endoplasmic reticulum (ko04141) and plant-pathogen interaction (ko04626) at the molecular level. SlDREBA4 also induces the expression of biosynthesis genes in jasmonic acid (JA), salicylic acid (SA), and ethylene (ETH), and specifically binds to the DRE elements (core sequence, A/GCCGAC) of the Hsp genes downstream from SlDREBA4. This study provides new genetic resources and rationales for tomato heat-tolerance breeding and the heat-related regulatory mechanisms of DREBs.

Resistance of genetically modified wheat plants, containing a double-stranded RNA suppressor of the

Aim. Analysis of physiological and biochemical characteristics and grain productivity traits of transgenic common wheat plants (Triticum aestivum L.) with a double-stranded RNA suppressor of the proline dehydrogenase gene (pdh) compared to non-transgenic genotypes under conditions of soil drought. Methods. Biochemical assays: spectrophotometric measurements of antioxidant enzymes (SOD, APX) activity, proline dehydrogenase activity and free L-proline content; biotechnological assays: Agrobacterium-mediated transformation in planta; physiological: morphometric traits and elements of grain productivity; mathematical statistics. Results. The presence of a double-stranded RNA suppressor of the gene pdh in transgenic plants leads not only to a decrease in enzyme activity (on average 2 times compared to the nontransgenic plants), but also to an increase in the content of free L-proline both under optimal conditions and under soil drought (2.6–4.1 times). Under soil moisture defi ciency, transgenic plants with reduced pdh gene activity in terms of yield structure signifi cantly exceeded the corresponding values of grain productivity elements for the non-transformed ones. In terms of the grain weight from the main stem, the biotechnological plants under soil moisture defi ciency exceeded the control plants 1.5–1.6 times, while the number of grains did not differ signifi cantly. The grain productivity of the whole plant in the transformed lines under conditions of soil moisture defi ciency was somewhat inferior to those in the nontransformed plants grown under optimal conditions. Soil drought caused a signifi cant increase in SOD and APX activity (by 40 and 11 %, respectively) in plants of the original variety. On the contrary, the activity of both antioxidant enzymes under drought conditions in transgenic plants decreased: SOD – down to 73–76 %, APX – down to 82–86 %, compared with the variant of 70 % of fi eld capacity. Conclusions. The analysis of physiological and biochemical characteristics, as well as economic and grain productivity elements of transgenic common wheat plants with a double-stranded RNA suppressor of the proline dehydrogenase gene showed their increased tolerance to soil drought, compared with non-transgenic genotypes, which may be associated with higher proline accumulation and an increase in the antioxidant enzymes activity. Under soil moisture defi ciency, transgenic wheat plants with reduced pdh gene activity signifi cantly exceeded the corresponding values of grain productivity elements for non-transformed plants. The analysis of the antioxidant enzymes activity in the chloroplasts of transgenic plants showed that under physiological conditions, the antioxidant system works more actively in comparison with non-transgenic genotypes, which may be a prerequisite for increasing the tolerance of these plants to the infl uence of stressors of various origin. It is likely that the positive relationship between the level of free L-proline and the resistance of transgenic wheat plants to osmotic stress is associated either with the effect of L-proline on the expression of other genes of the stress-strain response of plants, or with the positive effect of the increased content of this amino acid on resistance at the early stages of stress development. It has been suggested that an increase in the antioxidant enzymes activity in biotechnological plants can be caused by the expression of heterologous genes.

Expression of Tcchitinase-I gene in transgenic peanut (Arachis hypogaea L.) confers enhanced resistance against leaf spot and rust diseases

The disease-resistant transgenic peanut cv ICG 13942 plants were developed by using Tcchitinase-I gene. Agrobacterium tumefaciens strain LBA4404 harboring the binary vector (pBinAR) contains the chitinase (Tcchitinase-I) gene and neomycin phosphotransferase resistance (nptII) gene. The transformed shoots were developed on selection medium (MMS + 0.5 mg/L IAA + 15 mg/L TDZ + 100 mg/L Kan + 250 mg/L Cefotaxime) from deembryonated cotyledon (DC) explants. Established plantlets were screened for the presence of Tcchitinase-I and nptII genes. Stable integration and expression of the transgenes (T0) were confirmed by using PCR, RT-PCR and Southern blot analyses. The transformation frequency 63.34% was recorded. All the transformed (T0) plants were found normal, flowered and set seeds. After selfing the T0 plants, a Mendelian inheritance pattern (3:1) for the transgene in T1 progeny is revealed. T1 transgenic peanut plants were evaluated for resistance against Cercospora arachidicola, C. personatum and Puccinia arachidis by infection with the microspores using detached leaf assay. These T1 plants have shown longer incubation, latent period and lower infection frequencies in comparison to non-transformed (WT) plants. The Tcchitinase-I gene expression in resistant transgenic plants was compared to that of a susceptible control. A significant negative correlation was recorded between chitinase activity and the frequency of infection to the three tested disease causing agents.

MiR319c acts as a positive regulator of tomato against Botrytis cinerea infection by targeting TCP29

miR319 family is one of the oldest and most conservative miRNA families in plant and plays an important role in plant development and abiotic stress response. In our previous study, the abundance of sly-miR319c was increased in tomatoes infected by B. cinerea, but the roles and regulatory mechanisms of sly-miR319c in B. cinerea-infected tomato remain unclear. In this study, we confirmed that miR319c was increased in tomato with B. cinerea infection. In contrast, A TCP transcript factor, TCP29, targeted by sly-miR319c was decreased in B. cinerea-infected tomato. Therefore, transgenic Arabidopsis overexpressing sly-miR319c or its target were generated for understanding the biological roles and molecular mechanism of miR319c in B.cinerea-infected plants. Results showed that miR319c overexpression improved the resistance of transgenic plants to B. cinerea, whereas TCP29 overexpression increased the susceptibility of transgenic plant to B. cinerea. So far, TCP transcription factors have been reported mainly in developmental processes. Our data indicate that TCP29 act as a negative regulator to B.cinerea infection. In conclusion, our results indicate that sly-miR319c is a positive regulator of tomato resistance to B. cinerea infection by targeting TCP29.

Na+/H+ antiporter (NHX1) positively enhances cadmium (Cd) resistance and decreases Cd accumulation in tobacco plants cultivated in Cd-containing soil

Na+/H+ antiporter (NHX1) was reported to be induced by NaCl in plants and NaCl can alleviate the toxic effect in plants caused by cadmium (Cd). However, it is unknown whether the NHX1 gene is involved in Cd adaptation in plants. This study aimed to explore the relationship between Cd resistance and NHX1 in plants. SseNHX1 gene obtained by shuffling using SsNHX1 from Suaeda salsa and SeNHX1 from Salicornia europaea as sequence templates in our previous work, conferred higher salt tolerance to yeast than SsNHX1 and SeNHX1. The overexpression of SsNHX1, SeNHX1, or SseNHX1 genes in tobacco was performed to explore the relationship between Cd resistance and NHX1 gene expression in plants. The NHX1 inhibitor amiloride was also used to assess the relationship between Cd resistance and NHX1 in plants. Under Cd stress treatment, transgenic tobacco exhibited better growth status, increased chlorophyll content, improved photosynthesis capacity and enhanced content of Na+ and K+ compared to wildtype plants. Moreover, transgenic tobacco showed less ROS accumulation, reduced MDA production, and decreased Cd2+ accumulation compared to WT plants. More importantly, transgenic tobaccos overexpressing SseNHX1 gene showed a greater resistance to Cd stress and less Cd accumulation than the transgenic tobaccos overexpressing SsNHX1 or SeNHX1 genes. Cd resistance and Cd accumulation in transgenic tobacco plants was closely correlated with the expression of NHX1 gene.

An apple long-chain acyl-CoA synthetase 2 gene enhances plant resistance to abiotic stress by regulating the accumulation of cuticular wax.

Apple cuticular wax can protect plants from environmental stress, determine fruit luster and improve postharvest fruit storage quality. In recent years, dry weather, soil salinization and adverse environmental conditions have led to declines in apple fruit quality. However, few studies have reported the molecular mechanisms of apple cuticular wax biosynthesis. In this study, we identified a long-chain acyl-CoA synthetase MdLACS2 gene from apple. The MdLACS2 protein contained an AMP-binding domain and demonstrated long-chain acyl-CoA synthetase activity. MdLACS2 transgenic Arabidopsis exhibited reductions in epidermal permeability and water loss; change in the expression of genes related to cuticular wax biosynthesis, transport and transcriptional regulation; and differences in the composition and ultrastructure of cuticular wax. Moreover, the accumulation of cuticular wax enhanced the resistance of MdLACS2 transgenic plants to drought and salt stress. The main protein functional interaction networks of LACS2 were predicted, revealing a preliminary molecular regulation pathway for MdLACS2-mediated wax biosynthesis in apple. Our study provides candidate genes for breeding apple varieties and rootstocks with better fruit quality and higher stress resistance.