Enhanced resistance to sclerotinia stem rot in transgenic soybean that overexpresses a wheat oxalate oxidase

Sclerotinia stem rot (SSR), caused by the oxalate-secreting necrotrophic fungal pathogen Sclerotinia sclerotiorum, is one of the devastating diseases that causes significant yield loss in soybean (Glycine max). Until now, effective control of the pathogen is greatly limited by a lack of strong resistance in available commercial soybean cultivars. In this study, transgenic soybean plants overexpressing an oxalic acid (OA)-degrading oxalate oxidase gene OXO from wheat were generated and evaluated for their resistance to S. sclerotiorum. Integration and expression of the transgene were confirmed by Southern and western blot analyses. As compared with non-transformed (NT) control plants, the transgenic lines with increased oxalate oxidase activity displayed significantly reduced lesion sizes, i.e., by 58.71–82.73% reduction of lesion length in a detached stem assay (T₃ and T₄ generations) and 76.67–82.0% reduction of lesion area in a detached leaf assay (T₄ generation). The transgenic plants also showed increased tolerance to the externally applied OA (60 mM) relative to the NT controls. Consecutive resistance evaluation further confirmed an enhanced and stable resistance to S. sclerotiorum in the T₃ and T₄ transgenic lines. Similarly, decreased OA content and increased hydrogen peroxide (H₂O₂) levels were also observed in the transgenic leaves after S. sclerotiorum inoculation. Quantitative real-time polymerase chain reaction analysis revealed that the expression level of OXO reached a peak at 1 h and 4 h after inoculation with S. sclerotiorum. In parallel, a significant up-regulation of the hypersensitive response-related genes GmNPR1-1, GmNPR1-2, GmSGT1, and GmRAR occurred, eventually induced by increased release of H₂O₂ at the infection sites. Interestingly, other defense-related genes such as salicylic acid-dependent genes (GmPR1, GmPR2, GmPR3, GmPR5, GmPR12 and GmPAL), and ethylene/jasmonic acid-dependent genes (GmAOS, GmPPO) also exhibited higher expression levels in the transgenic plants than in the NT controls. Our results demonstrated that overexpression of OXO enhances SSR resistance by degrading OA secreted by S. sclerotiorum and increasing H₂O₂ levels, and eliciting defense responses mediated by multiple signaling pathways.

     

Salt tolerance conferred by expression of a global regulator IrrE from <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in oilseed rape

As salinity is a major threat to sustainable agriculture worldwide, cultivation of salt-tolerant crops becomes increasingly important. IrrE acts as a global regulator and a general switch for stress resistance in Deinococcus radiodurans. In this study, to determine whether the irrE gene can improve the salt tolerance of Brassica napus, we introduced the irrE gene into B. napus by the Agrobacterium tumefaciens-mediated transformation method. Forty-two independent transgenic plants were regenerated. Polymerase chain reaction (PCR) analyses confirmed that the irrE gene had integrated into the plant genome. Northern as well as Western blot analyses revealed that the transgene was expressed at various levels in transgenic plants. Analysis for the T₁ progenies derived from four independent transformants showed that irrE had enhanced the salt tolerance of T₁ in the presence of 350 mM NaCl. Furthermore, under salt stress, transgenic plants accumulated more compatible solutes (proline) and a lower level of malondialdehyde (MDA), and they had higher activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). However, agronomic traits were not affected by irrE gene overexpression in the transgenic B. napus plants. This study indicates that the irrE gene can improve the salt tolerance of B. napus and represents a promising candidate for the development of crops with enhanced salt tolerance by genetic engineering.     

Aspergillus spp. eliminate Sclerotinia sclerotiorum by imbalancing the ambient oxalic acid concentration and parasitizing its sclerotia

Sclerotinia sclerotiorum, a pathogen of more than 600 host plants, secretes oxalic acid to regulate the ambient acidity and provide conducive environment for pathogenicity and reproduction. Few Aspergillus spp. were previously proposed as potential biocontrol agents for S. sclerotiorum as they deteriorate sclerotia and prevent pathogen's overwintering and initial infections. We studied the nature of physical and biochemical interactions between Aspergillus and Sclerotinia. Aspergillus species inhibited sclerotial germination as they colonized its rind layer. However, Aspergillus-infested sclerotia remain solid and viable for vegetative and carpogenic germination, indicating that Aspergillus infestation is superficial. Aspergillus spp. of section Nigri (Aspergillus japonicus and Aspergillus niger) were also capable of suppressing sclerotial formation by S. sclerotiorum on agar plates. Their culture filtrate contained high levels of oxalic, citric and glutaric acids comparing to the other Aspergillus spp. tested. Exogenous supplementation of oxalic acid altered growth and reproduction of S. sclerotiorum at low concentrations. Inhibitory concentrations of oxalic acid displayed lower pH values comparing to their parallel concentrations of other organic acids. Thus, S. sclerotiorum growth and reproduction are sensitive to the ambient oxalic acid fluctuations and the environmental acidity. Together, Aspergillus species parasitize colonies of Sclerotinia and prevent sclerotial formation through their acidic secretions.     

Co-expression of chimeric chitinase and a polygalacturonase-inhibiting protein in transgenic canola <Emphasis Type="Italic">(Brassica napus)</Emphasis> confers enhanced resistance to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Objectives

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is one of the major fungal diseases of canola. To develop resistance against this fungal disease, the chit42 from Trichoderma atroviride with chitin-binding domain and polygalacturonase-inhibiting protein 2 (PG1P2) of Phaseolus vulgaris were co-expressed in canola via Agrobacterium-mediated transformation.

Results

Stable integration and expression of transgenes in T₀ and T₂ plants was confirmed by PCR, Southern blot and RT-PCR analyses. Chitinase activity and PGIP2 inhibition were detected by colorimetric and agarose diffusion assay in transgenic lines but not in untransformed plants. The crude proteins from single copy transformant leaves having high chitinase and PGIP2 activity (T16, T8 and T3), showed up to 44 % inhibition of S. sclerotiorum hyphal growth. The homozygous T₂ plants, showing inheritance in Mendelian fashion (3:1), were further evaluated under greenhouse conditions for resistance to S. sclerotiorum. Intact plants contaminated with mycelia showed resistance through delayed onset of the disease and restricted size and expansion of lesions as compared to wild type plants.

Conclusions

Combined expression of chimeric chit42 and pgip2 in Brassica napus L. provide subsequent protection against SSR disease and can be helpful in increasing the canola production in Iran.

     

Expressing a gene encoding wheat oxalate oxidase enhances resistance to Sclerotinia sclerotiorum in oilseed rape (Brassica napus)

Sclerotinia sclerotiorum causes a highly destructive disease in oilseed rape (Brassica napus). Oxalic acid (OA) secreted by the pathogen is a key pathogenicity factor. Oxalate oxidase (OXO) can oxidize OA into CO₂ and H₂O₂. In this study, we show that transgenic oilseed rape (sixth generation lines) constitutively expressing wheat (Triticum aestivum) OXO displays considerably increased OXO activity and enhanced resistance to S. sclerotiorum (with up to 90.2 and 88.4% disease reductions compared with the untransformed parent line and a resistant control, respectively). Upon application of exogenous OA, the pH values in transgenic plants were maintained at levels slightly lower than 5.58 measured prior to OA treatment, whereas the pH values in untransformed plants decreased rapidly and were markedly lower than 5.63 measured prior to OA treatment. Following pathogen inoculation, H₂O₂ levels were higher in transgenic plants than in untransformed plants. These results indicate that the enhanced resistance of the OXO transgenic oilseed rape to Sclerotinia is probably mediated by OA detoxification. We believe that enhancing the OA metabolism of oilseed rape in this way will be an effective strategy for improving resistance to S. sclerotiorum. Xiangbai Dong and Ruiqin Ji contributed equally to this paper.     

Co-transformation of canola by chimeric chitinase and <Emphasis Type="Italic">tlp</Emphasis> genes towards improving resistance to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Canola (Brassica napus) plants were co-transformed with two pathogenesis-related protein genes expressing a Trichoderma atroviride chitinase with a chitin-binding domain (chimeric chitinase) and a thaumatin-like protein (tlp) from Oryza sativa conferring resistance to phytopatogenic fungi by Agrobacterium-mediated transformation. The putative transgenic plants were confirmed by PCR. After measuring the specific activity of the chimeric chitinase and glucanase activity for tlp genes, transgenic plants with high specific activity were selected for southern blot analysis to confirm the copy number of the genes. In vitro assays, the antifungal activity of crude extracted protein against Sclerotinia sclerotiorum showed that the inhibition percentage in double transgenic plants was between 55 and 62, whereas the inhibition percentage in single-gene transformants (chimeric chitinase) ranged from 35 to 45 percent. Importantly, in greenhouse conditions, the double transgenic plants showed significant resistance than the single-gene transformant and wild type plants. The results in T₂ generation using the intact leaf inoculation method showed that the average lesion diameters were 10, 14.7 and 29 mm for the double transformant, single-gene transformant and non-transgenic plants, respectively. Combined expression of chimeric chitinase and tlp in transgenic plants showed significantly enhanced resistance against S. sclerotiorum than the one that express single-gene transformant plants. These results suggest that the co-expression of chimeric chitinase and tlp can confer enhanced disease resistance in canola plant.     

Resistance to Alternaria brassicicola in transgenic broccoli expressing a Trichoderma harzianum endochitinase gene

Transgenic broccoli plants expressing a Trichoderma harzianum endochitinase gene were obtained by Agrobacterium tumefaciens-mediated transformation. PCR and Southern blot analysis confirmed the presence of the gene in plants initially selected via resistance to kanamycin. Primary transformants (T₀) and selfed progeny (T₁) were examined for expression of the endochitinase gene using a fluorometric assay and for their resistance to the fungal pathogens Alternaria brassicicola and Sclerotinia sclerotiorum. All transgenic plants with elevated endochitinase activity had the expected 42 kDa endochitinase band in western blot analysis, whereas no such band was detected in the non-transgenic control. Leaves of most mature T₀ plants had 14–37 times higher endochitinase activity than controls; mature T₁ plants had higher endochitinase activity (100–200 times that in controls), in part because of lower control values. T₀ plantlets in vitro or young plants in soil had higher absolute and relative endochitinase activity. When detached leaves of T₀ plants were inoculated with A. brassicicola, lesion size showed a significant negative correlation with endochitinase levels. After inoculation of two-month old T₀ plants with A. brassicicola, all 15 transgenic lines tested showed significantly less severe disease symptoms than controls. In contrast, lesion size on petioles of T₀ and T₁ plants inoculated with S. sclerotiorum was not statistically different from controls.     

Pathogenicity Determinants of Sclerotinia sclerotiorum and Their Association to Its Aggressiveness on Brassica juncea

White rot or stem rot caused by Sclerotinia sclerotiorum is one of the most destructive fungal diseases that have become a serious threat to the successful cultivation of oilseed Brassicas. The study was designed with an aim to investigate the association between the pathogenic aggressiveness and pathogenicity determinants of this pathogen specifically in Brassica for the first time. For this, a total of 58 isolates of S. sclerotiorum from different geographical regions were collected and purified. These isolates were inoculated on a Brassica juncea cv. RL-1359 and they exhibited high level of variation in their disease progression. The isolates were grouped and then 24 isolates were selected for the biochemical analysis of pathogenicity determinants. The isolates varied significantly with respect to their total organic acids, oxalic acid production and pectin methyl esterase and polygalacturonase activity. The oxalic acid production corresponded to the disease progression of the isolates; the isolates with higher oxalic acid production were the more aggressive ones and vice-versa. This is, in our knowledge, the first study to establish a correlation between oxalic acid production and pathogenic aggressiveness of S. sclerotiorum on B. juncea. However, the pectinases’ enzyme activity did not follow the trend as of disease progression. These suggest an indispensable role of oxalic acid in pathogenicity of the fungus and the potential to be used as biochemical marker for preliminary assessment of pathogenic aggressiveness of various isolates before incorporating them in a breeding program.     

AtWRKY63基因过表达拟南芥对核盘菌抗性的研究

为研究草酸在核盘菌致病过程中可能的作用,以模式植物拟南芥为材料,采用30mmol/L草酸喷施3周龄拟南芥,发现草酸显著诱导拟南芥AtWRKY63的表达。通过构建AtWRKY63过表达载体转化拟南芥,获得过表达AtWRKY63的纯系转基因植株,再用核盘菌活体接种拟南芥,结果表明过表达AtWRKY63植株对核盘菌的抗性显著增强。组织化学染色结果表明,AtWRKY63是通过诱导植物的氧爆发,抑制核盘菌菌丝的生长来抵御核盘菌的侵染;qRT-PCR对拟南芥转录水平分析表明,AtWRKY63可能激活了过表达植株的水杨酸与茉莉酸依赖的抗病信号途径,从而增强对核盘菌的抗性。     

Differentially Expressed Proteins and Associated Histological and Disease Progression Changes in Cotyledon Tissue of a Resistant and Susceptible Genotype of Brassica napus Infected with Sclerotinia sclerotiorum

Sclerotinia rot caused by Sclerotinia sclerotiorum is one of the most serious diseases of oilseed rape. To understand the resistance mechanisms in the Brassica napus to S. sclerotiorum, comparative disease progression, histological and proteomic studies were conducted of two B. napus genotypes (resistant cv. Charlton, susceptible cv. RQ001-02M2). At 72 and 96 h post inoculation (hpi), lesion size on cotyledons was significantly (P≤0.001) smaller in the resistant Charlton. Anatomical investigations revealed impeded fungal growth (at 24 hpi and onwards) and hyphal disintegration only on resistant Charlton. Temporal changes (12, 24, 48 and 72 hpi) in protein profile showed certain enzymes up-regulated only in resistant Charlton, such as those related to primary metabolic pathways, antioxidant defence, ethylene biosynthesis, pathogenesis related proteins, protein synthesis and protein folding, play a role in mediating defence responses against S. sclerotiorum. Similarly a eukaryotic translation initiation factor 5A enzyme with increased abundance in susceptible RQ001-02M2 and decreased levels in resistant Charlton has a role in increased susceptibility to this pathogen. This is the first time that the expression of these enzymes has been shown to be associated with mediating the defence response against S. sclerotinia in cotyledon tissue of a resistant cultivar of B. napus at a proteomics level. This study not only provides important new insights into the resistance mechanisms within B. napus against S. sclerotiorum, but opens the way for novel engineering of new B. napus varieties that over-express these key enzymes as a strategy to enhance resistance and better manage this devastating pathogen.     

Transgenic <Emphasis Type="Italic">Brassica napus</Emphasis> L. lines carrying a two gene construct demonstrate enhanced resistance against <Emphasis Type="Italic">Plutella xylostella</Emphasis> and <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Plant diseases and insect pests are serious threat to the growth and yield of oilseed rape. In this study, a binary vector carrying sporamin and chitinase PjChi-1 genes in tandem was introduced into Brassica napus cv. ZS 758 via Agrobacterium tumefaciens for dual resistance against disease and insect attack. Thirty-two regenerated plantlets exhibiting hygromycin resistance were selected following Agrobacterium-mediated transformation of 600 leaf petiole explants. Of these, 27 transformants were confirmed to carry the two transgenes as detected by polymerase chain reaction (PCR) with 4.5% transformation efficiency. Eight plantlets were randomly selected for further confirmation by Southern and northern blot hybridization analyses. Four plants carried single copy of the transgenes, while the remaining four plants carried either two or three copies of the transgenes. Moreover, expression of the sporamin transgene was detected by northern blot hybridization in transgenic lines, but not in wild-type plants. These eight T₀ plants were grown in vitro, and inoculated with the Lepidoptera larvae of Plutella xylostella and with spores of the fungal pathogen of Sclerotinia sclerotiorum. Transgenic plants exhibited high levels of resistance to P. xylostella and S. sclerotiorum when compared to untransformed wild-type plants. Genetic analysis of T₁ progeny confirmed Mendelian segregation of the introduced genes. Therefore, these transgenic lines demonstrate a promising potential for variety development of oilseed rape lines with enhanced resistance against both P. xylostella and S. sclerotiorum.     

Assessment of inheritance pattern and agronomic performance of transgenic rapeseed having harpin<Subscript>Xooc</Subscript>-encoding <Emphasis Type="Italic">hrf2</Emphasis> Gene

hrf2 gene is a member of the harpin-encoding gene family of rice-pathogenic bacterium Xanthomonas oryzae pv. oryzicola. In our previous studies, we observed that harpin_Xooc could elicit hypersensitive cell death in non-host plants, induce disease and insect resistance in plants, and enhance plant growth. In this study, the rapeseed cultivar, Yangyou 4, was genetically engineered via Agrobacterium-mediated transformation to express the hrf2 gene. Polymerase chain reaction (PCR) and southern blot analyses of T₁ generation of transgenic rapeseed revealed stable integration and expression of the inserted gene hrf2. In addition, the resistance to Sclerotinia sclerotiorum was greatly enhanced. A comparison between agronomic characters of transgenic and control lines displayed significant differences in terms of plant height, stem width, number of pods per plant, number of seeds per pod, 1,000-seed weight, and seed yield per plant. Among lines with resistance to S. sclerotiorum, T₁1 had improved agronomic traits compared with controls with a 22.7% seed yield increase. These results suggest that the introduction of the hrf2 gene into rapeseed can be an effective strategy for enhancing resistance to S. sclerotiorum.     

铁蛋白基因表达对烟草耐低铁能力的影响

铁是植物生长发育的必需元素。由于土壤中的三价铁离子不能被植物直接利用。使一些植物经常表现出缺铁症状。为探讨利用铁蛋白基因提高植物耐低铁胁迫的作用,利用农杆菌介导法将大豆铁蛋白基因SoyFer1和内源反义铁蛋白基因NtFer2的cDNA分别导人烟草基因组,采集转基因烟草种子。对T1转基因烟草的卡那霉素抗性分析表明,整合到烟草基因组的外源基因多为单拷贝基因,也有少数为多拷贝基因。对具有卡那霉素抗性的转基因植株进行PCR检测和Northern杂交分析表明,外源基因已整合到烟草基因组中,并且得到了正确表达。将转基因株系移栽到铁离子浓度不同的培养基中生长2个月后进行比较表明,转大豆铁蛋白基因烟草株系的生长量明显高于非转基因烟草株系,而转内源反义铁蛋白基因烟草株系的生长量则明显低于非转基因烟草株系。转大豆铁蛋白基因和转内源反义铁蛋白基因烟草株系的叶绿素含量、丙二醛（MDA）含量和过氧化物酶（POD）活性等生理性状也发生了明显变化,表现为转大豆铁蛋白基因株系的叶绿素含量明显增加,POD活性明显增强,MDA含量明显降低：而转内源反义铁蛋白基因株系的叶绿素含量、POD活性和MDA含量等则表现为与转大豆铁蛋白基因株系的相反。铁蛋白过量表达提高了烟草耐低铁能力,而铁蛋白抑制表达则降低了烟草耐低铁能力。     

Effect of Seed Treatment by Cold Plasma on the Resistance of Tomato to Ralstonia solanacearum (Bacterial Wilt)

This study investigated the effect of cold plasma seed treatment on tomato bacterial wilt, caused by Ralstonia solanacearum (R. solanacearum), and the regulation of resistance mechanisms. The effect of cold plasma of 80W on seed germination, plant growth, nutrient uptake, disease severity, hydrogen peroxide (H₂O₂) concentration and activities of peroxidase (POD; EC 1.11.1.7), polyphenol oxidase (PPO; EC 1.10.3.2) and phenylalanine ammonia lyase (PAL; EC 4.3.1.5) were examined in tomato plants. Plasma treatment increased tomato resistance to R. solanacearum with an efficacy of 25.0%. Plasma treatment significantly increased both germination and plant growth in comparison with the control treatment, and plasma-treated plants absorbed more calcium and boron than the controls. In addition, H₂O₂ levels in treated plants rose faster and reached a higher peak, at 2.579 µM gFW⁻¹, 140% greater than that of the control. Activities of POD (421.3 U gFW⁻¹), PPO (508.8 U gFW⁻¹) and PAL (707.3 U gFW⁻¹) were also greater in the treated plants than in the controls (103.0 U gFW⁻¹, 166.0 U gFW⁻¹ and 309.4 U gFW⁻¹, respectively). These results suggest that plasma treatment affects the regulation of plant growth, H₂O₂ concentration, and POD, PPO and PAL activity in tomato, resulting in an improved resistance to R. solanacearum. Consequently, cold plasma seed treatment has the potential to control tomato bacterial wilt caused by R. solanacearum.     

铁蛋白基因表达对烟草耐低铁能力的影响

铁是植物生长发育的必需元素。由于土壤中的三价铁离子不能被植物直接利用, 使一些植物经常表现出缺铁症状。为探讨利用铁蛋白基因提高植物耐低铁胁迫的作用, 利用农杆菌介导法将大豆铁蛋白基因SoyFer1和内源反义铁蛋白基因NtFer2的cDNA分别导入烟草基因组, 采集转基因烟草种子。对T1转基因烟草的卡那霉素抗性分析表明, 整合到烟草基因组的外源基因多为单拷贝基因, 也有少数为多拷贝基因。对具有卡那霉素抗性的转基因植株进行PCR检测和Northern杂交分析表明, 外源基因已整合到烟草基因组中, 并且得到了正确表达。将转基因株系移栽到铁离子浓度不同的培养基中生长2个月后进行比较表明, 转大豆铁蛋白基因烟草株系的生长量明显高于非转基因烟草株系, 而转内源反义铁蛋白基因烟草株系的生长量则明显低于非转基因烟草株系。转大豆铁蛋白基因和转内源反义铁蛋白基因烟草株系的叶绿素含量、丙二醛(MDA)含量和过氧化物酶(POD)活性等生理性状也发生了明显变化, 表现为转大豆铁蛋白基因株系的叶绿素含量明显增加, POD活性明显增强, MDA含量明显降低; 而转内源反义铁蛋白基因株系的叶绿素含量、POD活性和MDA含量等则表现为与转大豆铁蛋白基因株系的相反。铁蛋白过量表达提高了烟草耐低铁能力, 而铁蛋白抑制表达则降低了烟草耐低铁能力。     

Prokaryotic expression and protein function of Brassica napus PGIP2 and its genetic transformation

Sclerotinia rot is a fungal disease caused by Sclerotinia sclerotiorum (Lib.) de Bary, which has severely reduced rapeseed production worldwide. Polygalacturonase-inhibiting proteins (PIGPs) inhibit the activity of polygalacturonases, which are secreted during fungal infection in plants. This study investigated the function of the polygalacturonase-inhibitor gene 2 (PGIP2) in sclerotinia rot resistance. The PGIP2 was successfully expressed in a prokaryotic system, and recombinant PGIP2 protein, purified after enterokinase treatment to remove tag peptide, inhibited S. sclerotiorum PG activity in vitro. PGIP2 was overexpressed in the susceptible Brassica napus cultivar 98c40 via Agrobacterium-mediated transformation. After inoculation with S. sclerotiorum mycelia, the transgenic rapeseed demonstrated greatly reduced leaf damage, as compared with their non-transgenic plants. Therefore, the PGIP2 encodes a functional protein and would be a candidate gene for enhancing Sclerotinia rot resistance.