Stable expression of exogenous imported <Emphasis Type="Italic">sporamin</Emphasis> in transgenic Chinese cabbage enhances resistance against insects

Cultivating insect pest-resistant varieties is one of the most effective ways to prevent or mitigate pest infestation in Chinese cabbage (Brassica campestris ssp. chinensis). Via the agrobacterium tumefaciens-mediated transformation method, this study introduced the protease inhibitor encoding gene sporamin into two widely cultured cultivars ‘Youdonger’ and ‘Shanghaiqing’, of the common variety of Chinese cabbages (B. campestriss ssp. chinensis var. communis), getting transgenic plants with high sporamin expression. In vitro insect bioassays indicated that, compared with the wild type plants, the transgenic plants exhibited improved resistance to diamondback moth (Plutella xylostella L.) The analysis of inheritance pattern of exogenous sporamin in the progenies of single copy insertion transgenic lines demonstrated that sporamin could be inherited and expressed stably in transgenic progenies. Field survey of the insect resistance under the normal culture condition confirmed the enhanced resistance in transgenic progenies to diamondback moth. Our results strongly suggest that sporamin is an efficient candidate gene for insect-resistant genetic engineering in Chinese cabbage.     

Effects of endogenous hormones on variation of shoot branching in a variety of non-heading Chinese cabbage and related gene expression

Shoot branching (tillering) primarily determines plant shoot architecture and has been studied in many plants. Shoot branching is an important trait in non-heading Chinese cabbage (Brassica rapa ssp. chinensis Makino). The B. rapa ssp. chinensis var. multiceps exhibits unique and multiple shoot branching characteristics. Here, we analyzed the variation in shoot branching between ‘Maertou,’ with multiple shoot branching, and ‘Suzhouqing,’ a common variety. The levels of endogenous indole-3-acetic acid (IAA), zeatin riboside and active gibberellins in the shoot meristem tissues of the two cultivars were quantified by enzyme-linked immunosorbent assay during the vegetative growth stage. High levels of IAA maintained axillary bud dormancy and repressed axillary bud outgrowth allowing shoot branching to form in the vegetative stage in ‘Suzhouqing.’ In contrast, low levels of IAA did not inhibit axillary buds in ‘Maertou,’ while a high level of cytokinin promoted axillary bud growth and branch shoot development. Exogenous hormone (rac-GR24 and 6-benzylaminopurine) treatment showed that ‘Maertou’ was relatively sensitive to cytokinin, because the fold changes of cytokinin-responsive genes in ‘Maertou’ were significantly more frequent than those in ‘Suzhouqing’. Cytokinin was the direct regulator for axillary bud growth of ‘Maertou’. Compared with ‘Suzhouqing’, ‘Maertou’ was sensitive to cytokinin and this weakened the strigolactone–cytokinin branching pathway.     

Pyr-miR171f-targeted <Emphasis Type="Italic">PyrSCL6</Emphasis> and <Emphasis Type="Italic">PyrSCL22</Emphasis> genes regulate shoot growth by responding to IAA signaling in pear

MicroRNA171 (miR171) is a highly conserved miRNA family, crucial for plant growth and development, and has been reported in Arabidopsis thaliana and tomato (Solanum lycopersicum), but the role of miR171 has not been explored in pear. In this study, an effort was made to decipher the mechanism underlying dwarf in ‘Zhongai 3’, of which the shoot length and shoot growth rate during the growing season were much less than those of the vigorous cultivar ‘Zaosu’, and the same for the indole-3-acetic acid (IAA) content in shoot tips after May 22, 2016. We identified a member of the miR171 family, which was most sensitive to IAA and targeted two genes conformed by 5′-RACE, and we named Pyr-miR171f. The two targets were named as PyrSCL6 and PyrSCL22, and contained a GRAS-conserved domain and encoded nucleus proteins. Quantitative RT-PCR analysis revealed that Pyr-miR171f was more abundant in ‘Zaosu’ shoot tips than in ‘Zhongai 3’ shoot tips, whereas the PyrSCL6 and PyrSCL22 mRNAs were more abundant in ‘Zhongai 3’ shoot tips than in ‘Zaosu’ shoot tips. The abundance of Pyr-miR171f and PyrSCL6 and PyrSCL22 mRNAs increased, but the trends were opposite between Pyr-miR171f and its target mRNAs in tissue culture seedlings treated by IAA. Our results suggest that IAA-induced miR171f negatively regulates the IAA signaling cascade via the GRAS pathway to maintain apical dominance. This work reveals a role for the miR171-SCL pathway in the dwarfing of ‘Zhongai 3’, and provides a theoretical basis for dwarf pear breeding.     

Effect of <Emphasis Type="Italic">Rol</Emphasis> Transgenes,IAA, and Kinetin on Starch Content and the Size of Starch Granules in Tubers of <Emphasis Type="Italic">In Vitro</Emphasis> Potato Plants

Stem cuttings were produced from Solanum tuberosum L., cv. Desiree, plants and their transgenic forms harboring rolB and rolC genes from Agrobacterium rhizogenes. Plants were cultured on hormone-free Murashige and Skoog nutrient medium (MS) and on MS supplemented with IAA or kinetin. In microtubers developed on these cuttings, we estimated the content of starch and the number and size of starch granules. Expression of rol genes changed these indices: in tubers of rolC transformants, a greater number of small granules were produced, whereas in tubers of rolB transformants, a fewer number of large granules were developed as compared with wild-type plants. Expression of rol genes did not affect starch content during the first three weeks of cutting culturing but increased it by 15–30% in five-week-old tubers. IAA addition to MS medium increased starch content and the size of starch granules in control plants and rolB tubers by 10–30%, whereas kinetin did not exert any significant influence. The effects of rol transgenes on the initiation and termination of starch granule development are discussed.     

A QTL detected in an interspecific pear population confers stable fire blight resistance across different environments and genetic backgrounds

Fire blight, caused by the bacterium Erwinia amylovora (Burrill) Winslow et al., is one of the most serious diseases of pear. The development of pear cultivars with a durable resistance is extremely important for effective control of fire blight and is a key objective of most pear breeding programs throughout the world. We phenotyped seedlings from the interspecific pear population PEAR3 (PremP003, P. × bretschneideri × P. communis) × ‘Moonglow’ (P. communis) for fire blight resistance at two different geographic locations, in France and New Zealand, respectively, employing two local E. amylovora isolates. Using a genetic map constructed with single nucleotide polymorphism (SNP) and microsatellite (SSR) markers previously developed for this segregating population, we detected a major quantitative trait locus (QTL) on linkage group (LG)2 of ‘Moonglow’ (R ² = 12.9–34.4 %), which was stable in both environments. We demonstrated that this QTL co-localizes with another major QTL for fire blight resistance previously detected in ‘Harrow Sweet’ and that the two favorable (i.e., resistant) alleles were not identical by descent. We also identified some smaller effect (R ² = 8.1–14.8 %) QTLs derived from the susceptible parent PEAR3. We propose SNP and SSR markers linked to the large effect QTL on LG2 as candidates for marker-assisted breeding for fire blight resistance in pear.     

Flooding events and rising water temperatures increase the significance of the reed pathogen <Emphasis Type="Italic">Pythium phragmitis</Emphasis> as a contributing factor in the decline of <Emphasis Type="Italic">Phragmites australis</Emphasis>

Pythium species are economically significant soilborne plant pathogens with worldwide distribution, causing seedling damping-off or root rot diseases. Pythium phragmitis is a newly described pathogen of common reed (Phragmites australis), widespread in the reed-belt of Lake Constance, Germany. It is highly aggressive towards reed leaves and seedlings, but obviously does not affect roots. In the context of ‘reed decline’ phenomena, P. phragmitis infection of reed inundated during flooding events may be of particular significance. We could show that flooding itself is not necessarily detrimental for reed plants. In the presence of the pathogen, however, most submerged leaves and plants were killed within several weeks. Clipped plants did not show regrowth in the Pythium infested treatments. Significant losses in assimilating leaf area of reeds could, thus, be the result of Pythium infection rather than of flooding alone. Therefore, we suggest that the combination of extended flooding and the presence of P. phragmitis might considerably contribute to ‘reed decline’ at Lake Constance. In parallel, we could show that pathogenicity and spread of this species are considerably favoured by rising temperatures. Since an increase in average water temperature has been found for Lake Constance, we propose that P. phragmitis could be an important factor in the dieback of reed stands likely to be promoted by predicted climate change phenomena.     

Attachment ability of the southern green stink bug, <Emphasis Type="Italic">Nezara viridula</Emphasis> (L.), on plant surfaces

Traction forces of male and female southern green stink bugs Nezara viridula (L.) were measured on adaxial leaves of green beans (Phaseolus vulgaris L. cv. ‘Nagauzura’), peas (Pisum sativum L. cv. ‘Hakuryu’), rice (Oryza sativa L. cv. ‘Mac hin sung’), and soybean (Glycine max [L.] Merr. cv. ‘Enrei’), as well as on glass as a control surface. Bugs attached well on three-dimensionally structured surfaces covered with anti-adhesive epicuticular wax crystals. Strongest pulls were generated on adaxial green bean leaves, corresponding to safety factors (traction force/body weight) of 11.1 and 11.6 in males and females, respectively. These values were slightly lower on soybean (males: 7.4, females: 8.0) and rice leaves (males: 8.9, females: 10.6). Trichomes and papillae are assumed to promote pentatomid bug’s attachment. On tabular, wax-covered pea leaves, safety factors decreased significantly to 1.7 and 1.6, in males and females, respectively. Differently, on non-structured glass, safety factors resembled those on rice and soybean leaves (males: 9.6, females: 8.0). No statistical differences in traction force and safety factor between sexes were detected on any substrate. Surface wettability did not significantly affect the results. Both robust claws and tough adhesive pads enable the N. viridula bugs to grasp and adhere to a wide range of various plant substrates, including such ones covered with anti-adhesive wax crystals. However, tabular, pruinose pea leaves were detected to prevent the foothold of the southern green stink bugs, although pea is known as one of their common host plants.     

The role of gibberellins and auxin on the tomato cell layers in pericarp via the expression of ARFs regulated by miRNAs in fruit set

Phytohormones, such as auxin (IAA) and gibberellin (GA), are known to be essential for fruit development. We utilized GA-deficient (gib-3) and diageotropica (dgt) tomato mutants to elucidate the effects of single hormones in the pericarp. The application of IAA or GA, respectively, to gib-3 or dgt single mutants induced a significant morphological difference in the fruit set. We found that IAA application induced cell division in the gib-3 pericarp and that GA application did not increase the cell layers in the dgt pericarp. In molecular studies, the expression levels of SlARF6, SlARF8, SlARF10 and SlARF16 were downregulated by IAA application, whereas the expression of their regulators miRNA160 and miRNA167 was upregulated by IAA application in gib-3 plants. Furthermore, the expression levels of SlARF6, SlARF8, SlARF10 and SlARF16 were upregulated by GA application, whereas the expression levels of miRNAs were reduced in the dgt mutant. These results imply that the expression levels of SlARF6, SlARF8, SlARF10 and SlARF16 were negatively correlated with the number of cell layers in the pericarp during fruit set. To further support this hypothesis, 35s:mSlARF10 transgenic plants resistant to SlmiR160 cleavage of SlARF10 mRNA were used to investigate the cell layers in fruit. These results revealed that mSlARF10 overexpression indeed resulted in fewer cell layers than in wild type fruit. Together, our data suggest that GA- and IAA-mediated miRNAs and their target ARFs influence the formation of pericarp cell layers during fruit set development.     

The <Emphasis Type="Italic">trans</Emphasis> and <Emphasis Type="Italic">cis</Emphasis> zeatin isomers play different roles in regulating growth inhibition induced by high nitrate concentrations in maize

Abscisic acid (ABA), auxins, and cytokinins (CKs) are known to be closely linked to nitrogen signaling. In particular, CKs control the effects of nitrate availability on plant growth. Our group has shown that treatment with high nitrate concentrations limits root growth and leaf development in maize, and conditions the development of younger roots and leaves. CKs also affect source-sink relationships in plants. Based on these results, we hypothesized that CKs regulate the source-sink relationship in maize via a mechanism involving complex crosstalk with the main auxin indole-3-acetic acid (IAA) and ABA. To evaluate this hypothesis, various CK metabolites, IAA, and ABA were quantified in the roots and in source and sink leaves of maize plants treated with high and normal nitrate concentrations. The data obtained suggest that the cis and trans isomers of zeatin play completely distinct roles in maize growth regulation by a complex crosstalk with IAA and ABA. We demonstrate that while trans-zeatin (tZ) and isopentenyladenine (iP) regulate nitrate uptake and thus control final leaf sizes, cis-zeatin (cZ) regulates source and sink strength, and thus controls leaf development. The implications of these findings relating to the roles of ABA and IAA in plants’ responses to varying nitrate concentrations are also discussed.     

Susceptibility of germinating cruciferous seeds to <Emphasis Type="Italic">Bagrada hilaris</Emphasis> (Hemiptera: Pentatomidae) feeding injury

Bagrada hilaris (Burmeister) (Hemiptera: Pentatomidae) is a serious pest that attacks both germinating and seedling stages of a variety of cruciferous crops grown in the Central Coast of California. B. hilaris feeding on germinating seeds can cause severe stunting and plant mortality, and little is known about the feeding preference of B. hilaris for germinating seeds of major cruciferous hosts and varieties of hosts. No-choice and choice experiments were conducted in which germinating seeds in soilless and soil settings were exposed to B. hilaris adults for 7 days. Susceptibility scores were developed using B. hilaris feeding injury sites, distorted leaves, and deformed and dead plants to determine the overall B. hilaris preference for germinating host seeds. Based on the scores, the order of preference was arugula (Eruca sativa L.)?>?turnip (B. rapa L. var. rapa)?>?mizuna (B. rapa L. nipposinica)?>?kale (B. oleracea L. acephala)?>?choi (Brassica rapa L. var. chinensis)?>?broccoli (B. oleracea var. italica Plenck)?>?cauliflower (B. oleracea L. var. botrytis)?>?lettuce (Lactuca sativa L.)?>?sweet alyssum (Lobularia maritima [L.] Desv.). The lowest feeding injury was recorded on germinating lettuce and sweet alyssum seeds. Furthermore, no-choice and choice experiments were conducted with four varieties each of arugula and mizuna, twelve varieties each of kale and choi, and nine varieties/types of leafy Asian greens. The arugula varieties ‘Wild Rocket’ and ‘Spirit’ were more damaged by B. hilaris than other varieties tested. Among mizuna varieties, ‘Beira F1’ was more attractive to B. hilaris than ‘Scarlet’ or ‘Starbor F1.’ The choi varieties ‘Tokyo Bekana,’ ‘Feng Qing Choi F1,’ ‘Joi Choi F1,’ and ‘Win-Win Choi F1’ were more attractive than ‘Rosie F1.’ The leafy Asian greens variety ‘Carlton F1’ was more attractive to B. hilaris than ‘Yukina Savon,’ ‘Tatsoi OG,’ ‘Komatsuna Summerfest F1,’ ‘Red Rain F1,’ and ‘Shungiku.’ Therefore, the results suggest that not all varieties were equally susceptible to B. hilaris feeding and possibly be utilized for further field evaluation as a trap crop or developing more resistant varieties to B. hilaris.     

Proteomics and functional analyses of <Emphasis Type="Italic">Arabidopsis</Emphasis> nitrilases involved in the defense response to microbial pathogens

Main conclusion

Proteomics and functional analyses of the Arabidopsis – Pseudomonas syringae pv. tomato interactions reveal that Arabidopsis nitrilases are required for plant defense and R gene-mediated resistant responses to microbial pathogens. A high-throughput in planta proteome screen has identified Arabidopsis nitrilase 2 (AtNIT2), which was de novo-induced by Pseudomonas syringae pv. tomato (Pst) infection. The AtNIT2, AtNIT3, and AtNIT4 genes, but not AtNIT1, were distinctly induced in Arabidopsis leaves by Pst infection. Notably, avirulent Pst DC3000 (avrRpt2) infection led to significant induction of AtNIT2 and AtNIT4 in leaves. Pst DC3000 and Pst DC3000 (avrRpt2) significantly grew well in leaves of nitrilase transgenic (nit2i-2) and mutant (nit1-1 and nit3-1) lines compared to the wild-type leaves. In contrast, NIT2 overexpression in nit2 mutants led to significantly high growth of the two Pst strains in leaves. The nitrilase transgenic and mutant lines exhibited enhanced susceptibility to Hyaloperonospora arabidopsidis infection. The nit2 mutation enhanced Pst DC3000 (avrRpt2) growth in salicylic acid (SA)-deficient NahG transgenic and sid2 and npr1 mutant lines. Infection with Pst DC3000 or Pst DC3000 (avrRpt2) induced lower levels of indole-3-acetic acid (IAA) in nit2i and nit2i NahG plants than in wild-type plants, but did not alter the IAA level in NahG transgenic plants. This suggests that Arabidopsis nitrilase 2 is involved in IAA signaling of defense and R gene-mediated resistance responses to Pst infection. Quantification of SA in these transgenic and mutant plants demonstrates that Arabidopsis nitrilase 2 is not required for SA-mediated defense response to the virulent Pst DC3000 but regulates SA-mediated resistance to the avirulent Pst DC3000 (avrRpt2). These results collectively suggest that Arabidopsis nitrilase genes are involved in plant defense and R gene-mediated resistant responses to microbial pathogens.

     

Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice

Genetic engineering approaches offer an alternative method to the conventional breeding of Citrus sp. ‘W. Murcott’ mandarin (a hybrid of ‘Murcott’ and an unknown pollen parent) is one of the most commercially important cultivars grown in many regions around the world. Transformation of ‘W. Murcott’ mandarin was achieved by direct DNA uptake using a protoplast transformation system. DNA construct (pAO3), encoding Green Fluorescent Protein (GFP) and the cDNA of Xa21, a Xanthomonas resistance gene from rice, was used to transform protoplasts of ‘W. Murcott’ mandarin. Following citrus protoplast culture and regeneration, transformed micro calli were microscopically designated via GFP expression, physically isolated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. More than 150 transgenic embryos were recovered and from them, ten transgenic lines were regenerated and cultured on rooting medium for shoot elongation. Transgenic shoots were micrografted and established in the greenhouse with 3–5 replicates per line. The insertion of Xa21 and GFP was confirmed by PCR and southern blot analysis. GFP expression was verified by fluorescence microscopy and western blot analysis revealed expression of Xa21 although it was variable among transgenic lines, as shown by RT-qPCR. Transgenic plants challenged with the citrus canker pathogen by syringe inoculation showed a reduction in lesion number and bacterial populations within lesions compared to non-transgenic control plants. Transgenic ‘W. Murcott’ mandarin lines with improved canker resistance via protoplast transformation from embryogenic callus with the Xa21 gene from rice are being evaluated under field conditions to validate the level of resistance.     

Characterization of <Emphasis Type="Italic">Lr75</Emphasis>: a partial,broad-spectrum leaf rust resistance gene in wheat

Key message

Here, we describe a strategy to improve broad-spectrum leaf rust resistance by marker-assisted combination of two partial resistance genes. One of them represents a novel partial adult plant resistance gene, named Lr75.

Abstract

Leaf rust caused by the fungal pathogen Puccinia triticina is a damaging disease of wheat (Triticum aestivum L.). The combination of several, additively-acting partial disease resistance genes has been proposed as a suitable strategy to breed wheat cultivars with high levels of durable field resistance. The Swiss winter wheat cultivar ‘Forno’ continues to show near-immunity to leaf rust since its release in the 1980s. This resistance is conferred by the presence of at least six quantitative trait loci (QTL), one of which is associated with the morphological trait leaf tip necrosis. Here, we used a marker-informed strategy to introgress two ‘Forno’ QTLs into the leaf rust-susceptible Swiss winter wheat cultivar ‘Arina’. The resulting backcross line ‘ArinaLrFor’ showed markedly increased leaf rust resistance in multiple locations over several years. One of the introgressed QTLs, QLr.sfr-1BS, is located on chromosome 1BS. We developed chromosome 1B-specific microsatellite markers by exploiting the Illumina survey sequences of wheat cv. ‘Chinese Spring’ and mapped QLr.sfr-1BS to a 4.3 cM interval flanked by the SSR markers gwm604 and swm271. QLr.sfr-1BS does not share a genetic location with any of the described leaf rust resistance genes present on chromosome 1B. Therefore, QLr.sfr-1BS is novel and was designated as Lr75. We conclude that marker-assisted combination of partial resistance genes is a feasible strategy to increase broad-spectrum leaf rust resistance. The identification of Lr75 adds a novel and highly useful gene to the small set of known partial, adult plant leaf rust resistance genes.

     

Adventitious shoot regeneration from in vitro leaf explants of <Emphasis Type="Italic">Fraxinus nigra</Emphasis>

In order to establish an attractive method for the production of valuable medicinal alkaloids (galanthamine and lycorine), the plants of Leucojum aestivum and L. aestivum ‘Gravety Giant’ grown in bioreactor RITA® were subjected to various concentrations of methyl jasmonate (MeJA), salicylic acid (SA), 1-aminocyclopropane-1-carboxylic acid (ACC) and 2-chloroethylphosphonic acid (ethephon) at different times of culture. The application of MeJA showed a negative effect on L. aestivum and L. aestivum ‘Gravety Giant’ plant growth. We observed that the incubation of plants during 168 h with 100 µM of MeJA resulted above two times lower F.W. (fresh weight) increments compared with control. While SA showed an inhibitory effect only on the growth of L. aestivum cultures. ACC and ethephon had a positive effect on both types of culture. Treatment with 50 µM of MeJA during 168 h stimulated galanthamine and lycorine biosynthesis in L. aestivum and L. aestivum ‘Gravety Giant’ cultures. In addition, the accumulation of galanthamine was increased when 10 µM of ACC were added to both types of culture. 10 µM of ACC stimulated also lycorine biosynthesis by L. aestivum ‘Gravety Giant’. The addition of 10 µM of ethephon had a positive effect only on lycorine production in plants of L. aestivum. SA promoted galanthamine and lycorine biosynthesis in tested plants. Indeed the highest galanthamine (0.8 mg/g dry weight: D.W.) and lycorine (1.53 mg/g D.W.) concentrations were observed in L. aestivum ‘Gravety Giant’ plants treated with 5 µM of SA during 10 h.