Overexpression of AtPAD4 in transgenic Brachypodium distachyon enhances resistance to Puccinia brachypodii

• Brachypodium distachyon (L.) has recently emerged as a model for temperate grasses for investigating the molecular basis of plant–pathogen interactions. Phytoalexin deficient 4 (PAD4) plays a regulatory role in mediating expression of genes involved in plant defence.
• In this research, we generated transgenic B. distachyon plants constitutively overexpressing AtPAD4. Two transgenic B. distachyon lines were verified using PCR and GUS phenotype.
• Constitutive expression of AtPAD4 in B. distachyon enhanced resistance to Puccinia brachypodii. P. brachypodii generated less urediniospores on transgenic than on wild‐type plants. AtPAD4 overexpression enhanced salicylic acid (SA) levels in B. distachyon‐infected tissues. qRT‐PCR showed that expression of pathogenesis‐related 1 (PR1) and other defence‐related genes were up‐regulated in transformed B. distachyon following infection with P. brachypodii.
• Our results indicate that AtPAD4 overexpression in B. distachyon plants led to SA accumulation and induced PR gene expression that reduced the rate of colonisation by P. brachypodii.

     

Osmotic shock improves <Emphasis Type="Italic">Tnt1</Emphasis> transposition frequency in <Emphasis Type="Italic">Medicago truncatula</Emphasis> cv Jemalong during in vitro regeneration

Insertion mutant collections are powerful tools for genetic studies in plants. Although large-scale insertional mutagenesis using T-DNA is not feasible in legumes, the Tnt1 tobacco retrotransposon can be used as a very efficient mutagen in the Medicago truncatula R108 genotype. In this article, we show that Tnt1 can also be exploited to create insertional mutants via transformation and/or regeneration in the reference cultivar Jemalong. Tnt1 insertional mutagenesis in Jemalong following Agrobacterium tumefaciens-mediated transformation was found to be very efficient, with an average of greater than 15 insertions/line. In contrast, regeneration using low-copy transgenic starter lines resulted in a highly variable rate of new Tnt1 insertions. With the goal of increasing the number of additional Tnt1 insertions during regeneration of starter lines, we have compared the insertion frequencies for a number of different regeneration protocols. In addition, we have been able to show that sucrose-mediated osmotic shock preceding regeneration significantly increases the transposition frequency. Under optimal conditions, 95% of the regenerated Jemalong plants possess new insertions.     

Sequencing and functional validation of the JGI Brachypodium distachyon T‐DNA collection

Due to a large and growing collection of genomic and experimental resources, Brachypodium distachyon has emerged as a powerful experimental model for the grasses. To add to these resources we sequenced 21 165 T‐DNA lines, 15 569 of which were produced in this study. This increased the number of unique insertion sites in the T‐DNA collection by 21 078, bringing the overall total to 26 112. Thirty‐seven per cent (9754) of these insertion sites are within genes (including untranslated regions and introns) and 28% (7217) are within 500 bp of a gene. Approximately 31% of the genes in the v.2.1 annotation have been tagged in this population. To demonstrate the utility of this collection, we phenotypically characterized six T‐DNA lines with insertions in genes previously shown in other systems to be involved in cellulose biosynthesis, hemicellulose biosynthesis, secondary cell wall development, DNA damage repair, wax biosynthesis and chloroplast synthesis. In all cases, the phenotypes observed supported previous studies, demonstrating the utility of this collection for plant functional genomics. The Brachypodium T‐DNA collection can be accessed at http://jgi.doe.gov/our-science/science-programs/plant-genomics/brachypodium/brachypodium-t-dna-collection/ .     

Somatic Hybrids Between Potato and S. berthaultii Show Partial Resistance to Soil‐Borne Fungi and Potato Virus Y

Three tetraploid somatic hybrid lines produced by protoplast fusion between a dihaploid potato, Solanum tuberosum, cultivar BF15 and the wild potato species Solanum berthaultii were evaluated here for their response to different soil‐borne pathogens, that is Fusarium solani, Pythium aphanidermatum and Rhizoctonia solani as well as to infection by potato virus Y (PVY). Both hybrid and BF15 plants grown in vitro were inoculated with the tested pathogen strains, that is R. solani, P. aphanidermatum, or F. solani. The growth level and disease severity index of these plants were compared to the susceptible commercial cultivar Spunta. A better growth of inoculated hybrid plants and restricted disease symptoms were observed in comparison with the commercial plants. Under glasshouse conditions and after inoculation with R. solani and P. aphanidermatum, improved resistance of the hybrid plants to these pathogens was confirmed. Indeed, these plants showed no significant damage following inoculation and a better development in R. solani‐infected plants. The susceptibility of the hybrid tubers to R. solani, P. aphanidermatum, and to F. solani infection was also determined. A significant reduction of tissue colonisation was observed in all the hybrid lines compared to the cultivated cultivars. The STBc and STBd hybrids also showed improved resistance to the PVY ordinary strain (PVY^o) under glasshouse conditions.     

Genetic mapping shows intraspecific variation and transgressive segregation for caterpillar‐induced aphid resistance in maize

Plants in nature have inducible defences that sometimes lead to targeted resistance against particular herbivores, but susceptibility to others. The metabolic diversity and genetic resources available for maize (Zea mays) make this a suitable system for a mechanistic study of within‐species variation in such plant‐mediated interactions between herbivores. Beet armyworms (Spodoptera exigua) and corn leaf aphids (Rhopalosiphum maidis) are two naturally occurring maize herbivores with different feeding habits. Whereas chewing herbivore‐induced methylation of 2,4‐dihydroxy‐7‐methoxy‐1,4‐benzoxazin‐3‐one glucoside (DIMBOA‐Glc) to form 2‐hydroxy‐4,7‐dimethoxy‐1,4‐benzoxazin‐3‐one glucoside (HDMBOA‐Glc) promotes caterpillar resistance, lower DIMBOA‐Glc levels favour aphid reproduction. Thus, caterpillar‐induced DIMBOA‐Glc methyltransferase activity in maize is predicted to promote aphid growth. To test this hypothesis, the impact of S. exigua feeding on R. maidis progeny production was assessed using seventeen genetically diverse maize inbred lines. Whereas aphid progeny production was increased by prior caterpillar feeding on lines B73, Ki11, Ki3 and Tx303, it decreased on lines Ky21, CML103, Mo18W and W22. Genetic mapping of this trait in a population of B73 × Ky21 recombinant inbred lines identified significant quantitative trait loci on maize chromosomes 1, 7 and 10. There is a transgressive segregation for aphid resistance, with the Ky21 alleles on chromosomes 1 and 7 and the B73 allele on chromosome 10 increasing aphid progeny production. The chromosome 1 QTL coincides with a cluster of three maize genes encoding benzoxazinoid O‐methyltransferases that convert DIMBOA‐Glc to HDMBOA‐Glc. Gene expression studies and benzoxazinoid measurements indicate that S. exigua ‐induced responses in this pathway differentially affect R. maidis resistance in B73 and Ky21.     

Impaired sterol ester synthesis alters the response of Arabidopsis thaliana to Phytophthora infestans

Non‐host resistance of Arabidopsis thaliana against Phytophthora infestans, the causal agent of late blight disease of potato, depends on efficient extracellular pre‐ and post‐invasive resistance responses. Pre‐invasive resistance against P. infestans requires the myrosinase PEN2. To identify additional genes involved in non‐host resistance to P. infestans, a genetic screen was performed by re‐mutagenesis of pen2 plants. Fourteen independent mutants were isolated that displayed an enhanced response to Phytophthora (erp) phenotype. Upon inoculation with P. infestans, two mutants, pen2‐1 erp1‐3 and pen2‐1 erp1‐4, showed an enhanced rate of mesophyll cell death and produced excessive callose deposits in the mesophyll cell layer. ERP1 encodes a phospholipid:sterol acyltransferase (PSAT1) that catalyzes the formation of sterol esters. Consistent with this, the tested T‐DNA insertion lines of PSAT1 are phenocopies of erp1 plants. Sterol ester levels are highly reduced in all erp1/psat1 mutants, whereas sterol glycoside levels are increased twofold. Excessive callose deposition occurred independently of PMR4/GSL5 activity, a known pathogen‐inducible callose synthase. A similar formation of aberrant callose deposits was triggered by the inoculation of erp1 psat1 plants with powdery mildew. These results suggest a role for sterol conjugates in cell non‐autonomous defense responses against invasive filamentous pathogens.     

Inoculation of susceptible and resistant potato plants with the late blight pathogen Phytophthora infestans: effects on an aphid and its parasitoid

Plants are exposed to microbial pathogens as well as herbivorous insects and their natural enemies. Here, we examined the effects of inoculation of potato plants, Solanum tuberosum L. (Solanaceae), with the late blight pathogen Phytophthora infestans (Mont.) de Bary (Peronosporales: Pythiaceae) on an aphid species commonly infesting potato crops and one of the aphid's major parasitoids. We observed the peach‐potato aphid, Myzus persicae Sulzer (Hemiptera: Aphididae), and its natural enemy, the biocontrol agent Aphidius colemani Viereck (Hymenoptera: Braconidae), on potato either inoculated with water or P. infestans. Population growth of the aphid, parasitism rate of its natural enemy, and other insect life‐history traits were compared on several potato genotypes, the susceptible cultivar Désirée and genetically modified (GM) isogenic lines carrying genes conferring resistance to P. infestans. Effects of P. infestans inoculation on the intrinsic rate of aphid population increase and the performance of the parasitoid were only found on the susceptible cultivar. Insect traits were similar when comparing inoculated with non‐inoculated resistant GM genotypes. We also tested how GM‐plant characteristics such as location of gene insertion and number of R genes could influence non‐target insects by comparing insect performance among GM events. Different transformation events leading to different positions of R‐gene insertion in the genome influenced aphids either with or without P. infestans infection, whereas effects of position of R‐gene insertion on the parasitoid A. colemani were evident only in the presence of inoculation with P. infestans. We conclude that it is important to study different transformation events before continuing with further stages of risk assessment of this GM crop. This provides important information on the effects of plant resistance to a phytopathogen on non‐target insects at various trophic levels.     

A dual gene‐silencing vector system for monocot and dicot plants

Plant virus‐based gene‐silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus‐induced gene‐silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene‐silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N. benthamiana. GFP transgenic plants co‐agro‐inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat‐shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV‐ but not BaMV‐based vector could enhance gene‐silencing efficiency in newly emerging leaves of N. benthamiana deficient in RNA‐dependant RNA polymerase 6. The dual gene‐silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.     

Engineering linear,branched‐chain triterpene metabolism in monocots

Triterpenes are thirty‐carbon compounds derived from the universal five‐carbon prenyl precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Normally, triterpenes are synthesized via the mevalonate (MVA) pathway operating in the cytoplasm of eukaryotes where DMAPP is condensed with two IPPs to yield farnesyl diphosphate (FPP), catalyzed by FPP synthase (FPS). Squalene synthase (SQS) condenses two molecules of FPP to generate the symmetrical product squalene, the first committed precursor to sterols and most other triterpenes. In the green algae Botryococcus braunii, two FPP molecules can also be condensed in an asymmetric manner yielding the more highly branched triterpene, botryococcene. Botryococcene is an attractive molecule because of its potential as a biofuel and petrochemical feedstock. Because B. braunii, the only native host for botryococcene biosynthesis, is difficult to grow, there have been efforts to move botryococcene biosynthesis into organisms more amenable to large‐scale production. Here, we report the genetic engineering of the model monocot, Brachypodium distachyon, for botryococcene biosynthesis and accumulation. A subcellular targeting strategy was used, directing the enzymes (botryococcene synthase [BS] and FPS) to either the cytosol or the plastid. High titres of botryococcene (>1 mg/g FW in T₀ mature plants) were obtained using the cytosolic‐targeting strategy. Plastid‐targeted BS + FPS lines accumulated botryococcene (albeit in lesser amounts than the cytosolic BS + FPS lines), but they showed a detrimental phenotype dependent on plastid‐targeted FPS, and could not proliferate and survive to set seed under phototrophic conditions. These results highlight intriguing differences in isoprenoid metabolism between dicots and monocots.     

Interactions Between Rhizoctonia Root Rot and the Foliar Common Bean Diseases Anthracnose and Rust

The effects of co‐inoculation of Rhizoctonia solani and Colletotrichum lindemuthianum or Uromyces appendiculatus at different inoculum levels were studied on the disease dynamics and on the growth of bean plants under greenhouse conditions. Bean seeds were sown in R. solani‐infested soil. Additional experiments in which seedlings were transplanted to infested soil were also carried out. Conidial suspensions of C. lindemuthianum or uredospores of U. appendiculatus were inoculated onto leaves at plant developmental stages V2 and V3, respectively. Interactions between root rot and the aerial diseases were observed depending on the inoculum levels and on the timing of R. solani inoculation. Anthracnose severity tended to be higher on R. solani‐infected plants. Conversely, R. solani infection significantly reduced diameter of pustules and rust severity. When seedlings were transplanted to soil infested with low levels of R. solani, root rot severity and density of R. solani in the soil were magnified at high levels of C. lindemuthianum or U. appendiculatus. In these experiments, a synergistic interaction between root rot and anthracnose was observed to affect the plant dry weight. Antagonistic effects on the plant dry weight were found for the combination root rot/rust only when seeds were sown in infested soil.     

Resistance to Asparagus virus 1 in the Wild Relative Asparagus amarus

Five asparagus cultivars, three breeding lines and the wild relative Asparagus amarus were tested for natural infection by Asparagus virus 1 (AV‐1) in experimental fields at two locations over 3 and 4 years, respectively. In the first year after re‐planting the annual crowns in the field, more than 90% of tested plants of cultivars were infected by AV‐1. In the third and fourth year, 100% of tested plants of cultivars were AV‐1 infected. In comparison, all plants of the wild relative A. amarus were completely free of AV‐1, suggesting a high level of resistance. Additionally, 1‐year‐old glasshouse‐cultivated plants of A. officinalis and A. amarus were placed in an AV‐1 provocation cabin under field conditions. Seven months later, 100% of the A. officinalis plants showed a high virus concentration in ELISA, whereas no AV‐1 was detectable in the A. amarus plants. This result was confirmed by highly sensitive AV‐1‐specific RT‐PCR. To exclude vector resistance, the feeding behaviour of green peach aphid Myzus persicae was tested over 12 h using the electrical penetration graph method. Both asparagus genotypes were accepted by the aphids as potential hosts, but the feeding time was significantly longer on A. amarus. A genetic distance analysis of the various cultivars of Asparagus officinalis and selected wild relatives of the JKI collection was carried out, resulting in a clear discrimination of cultivars and wild relatives, especially A. amarus. The potential breeding value of the putative resistance carrier is discussed.     

Identification and Pathogenicity of Lasiodiplodia Species from Eucalyptus urophylla × grandis,Polyscias balfouriana and Bougainvillea spectabilis in Southern China

Species of Lasiodiplodia are important pathogens of a wide variety of plants covering a wide geographical distribution. These fungi can be associated with different symptoms such as stem cankers, shoot blights, fruit rots, dieback and gummosis. Diseases caused by Lasiodiplodia were surveyed on Eucalyptus urophylla × grandis, Polyscias balfouriana and Bougainvillea spectabilis in a nursery in southern China. Based on morphology characteristics and phylogenetic analyses of ITS rDNA sequences and translation elongation factor 1‐alpha (TEF‐1α) gene regions, four species of Lasiodiplodia were identified. Lasiodiplodia theobromae was identified from E. urophylla × grandis, P. balfouriana and B. spectabilis. L. hormozganensis, L. iraniensis and L. pseudotheobromae were identified from B. spectabilis. To our knowledge, with the exception of L. theobromae on E. urophylla × grandis, this study represents the first report of these fungi on the host plants. Pathogenicity tests showed that all Lasiodiplodia spp. obtained in this study are virulent to E. urophylla × grandis and B. spectabilis, and L. theobromae was virulent to P. balfouriana.     

Mixed Infection and Natural Spread of ‘Candidatus Phytoplasma asteris’ and Mungbean Yellow Mosaic India Virus Affecting Soya Bean Crop in India

Suspected phytoplasma and virus‐like symptoms of little leaf, yellow mosaic and witches’ broom were recorded on soya bean and two weed species (Digitaria sanguinalis and Parthenium hysterophorus), at experimental fields of Indian Agricultural Research Institute, New Delhi, India, in August–September 2013. The phytoplasma aetiology was confirmed in symptomatic soya bean and both the weed species by direct and nested PCR assays with phytoplasma‐specific universal primer pairs (P1/P6 and R16F2n/R16R2n). One major leafhopper species viz. Empoasca motti Pruthi feeding on symptomatic soya bean plants was also found phytoplasma positive in nested PCR assays. Sequencing BLASTn search analysis and phylogenetic analysis revealed that 16Sr DNA sequences of phytoplasma isolates of soya bean, weeds and leafhoppers had 99% sequence identity among themselves and were related to strains of ‘Candidatus Phytoplasma asteris’. PCR assays with Mungbean yellow mosaic India virus (MYMIV) coat‐protein‐specific primers yielded an amplicon of approximately 770 bp both from symptomatic soya bean and from whiteflies (Bemisia tabaci) feeding on soya bean, confirmed the presence of MYMIV in soya bean and whitefly. Hence, this study suggested the mixed infection of MYMIV and ‘Ca. P. asteris’ with soya bean yellow leaf and witches’ broom syndrome. The two weed species (D. sanguinalis and P. hysterophorus) were recorded as putative alternative hosts for ‘Ca. P. asteris’ soya bean Indian strain. However, the leafhopper E. motti was recorded as putative vector for the identified soya bean phytoplasma isolate, and the whitefly (B. tabaci) was identified as vector of MYMIV which belonged to Asia‐II‐1 genotype.     

Effects of Tomato chlorosis virus on the performance of its key vector,Bemisia tabaci,in China

Tomato chlorosis virus (ToCV), which is a newly emerged and rapidly spreading plant virus in China, has seriously reduced tomato production and quality over the past several years. In this study, the effect of ToCV on the demography of the whitefly, Bemisia tabaci biotype Q (Hemiptera: Aleyrodidae), fed on infected and healthy tomato plants was evaluated using the age‐stage, two‐sex life table. When reared on ToCV‐infected tomato plants, the fecundity, length of oviposition period and female adult longevity of B. tabaci biotype Q decreased significantly, while the pre‐adult duration significantly increased compared to controls reared on healthy tomatoes. Consequently, the intrinsic rate of increase (r) and finite of increase (λ) of B. tabaci biotype Q on ToCV‐infected tomato plants significantly decreased compared to those on healthy tomatoes. Population projection predicted that a population of B. tabaci biotype Q fed on ToCV‐infected tomatoes increases slower than on healthy plants. These findings demonstrated that ToCV infection decreased the performance of B. tabaci biotype Q on tomato plants.