Completion of the sexual cycle and demonstration of genetic recombination in Ustilago maydis in vitro

The heterobasidiomycetes responsible for plant smuts obligatorily require their hosts for the completion of the sexual cycle. Accordingly, the sexual cycle of these fungi could so far be studied only by infecting host plants. We have now induced Ustilago maydis, the causative agent of corn smut, to traverse the whole life cycle by growing mixtures of mating-compatible strains of the fungus on a porous membrane placed on top of embryogenic cell cultures of its host Zea mays. Under these conditions, mating, karyogamy and meiosis take place, and the fungus induces differentiation of the plant cells. These results suggest that embryogenic maize cells produce diffusible compounds needed for completion of the sexual cycle of U. maydis, as the plant does for the pathogen during infection. Received: 16 February 1999 / Accepted: 30 June 1999     

The core effector Cce1 is required for early infection of maize by Ustilago maydis

The biotrophic pathogen Ustilago maydis, the causative agent of corn smut disease, infects one of the most important crops worldwide – Zea mays. To successfully colonize its host, U. maydis secretes proteins, known as effectors, that suppress plant defense responses and facilitate the establishment of biotrophy. In this work, we describe the U. maydis effector protein Cce1. Cce1 is essential for virulence and is upregulated during infection. Through microscopic analysis and in vitro assays, we show that Cce1 is secreted from hyphae during filamentous growth of the fungus. Strikingly, Δcce1 mutants are blocked at early stages of infection and induce callose deposition as a plant defense response. Cce1 is highly conserved among smut fungi and the Ustilago bromivora ortholog complemented the virulence defect of the SG200Δcce1 deletion strain. These data indicate that Cce1 is a core effector with apoplastic localization that is essential for U. maydis to infect its host.     

Influence of carbon and nitrogen concentration on itaconic acid production by the smut fungus Ustilago maydis

Itaconic acid is a valuable platform compound for the production of bio‐based polymers, chemicals, and fuels. Ustilago maydis is a promising host for the production of itaconic acid from biomass‐derived substrates due to its unicellular growth pattern and its potential to utilize biomass‐derived sugar monomers and polymers. The potential of U. maydis for industrial itaconate production was assessed in pH‐controlled batch fermentations with varying medium compositions. Using 200 g/L glucose and 75 mM ammonium, 44.5 g/L of itaconate was produced at a maximum rate of 0.74 g L^?1 h^?1. By decreasing the substrate concentrations to 50 g/L glucose and 30 mM ammonium, a yield of 0.34 g/g (47 mol%) could be achieved. Itaconate production from xylose was also feasible. These results indicate that high itaconic acid titers can be achieved with U. maydis. However, further optimization of the biocatalyst itself through metabolic engineering is still needed in order to achieve an economically feasible process, which can be used to advance the development of a bio‐based economy.     

Biological origins of normal-chain hydrocarbons: a pathway model based on cuticular wax analyses of maize silks

Long-chain normal hydrocarbons (e.g. alkanes, alkenes and dienes) are rare biological molecules and their biosynthetic origins are obscure. Detailed analyses of the surface lipids that accumulate on maize silks have revealed that these hydrocarbons constitute a large portion (>90%) of the cuticular waxes that coat this organ, which contrasts with the situation on maize seedling leaves, where the cuticular waxes are primary alcohols and aldehydes. The normal hydrocarbons that occur on silks are part of a homologous series of alkanes, alkenes and dienes of odd-number carbon atoms, ranging between 19 and 33 in number. The alkenes and dienes consist of a homologous series, each of which has double bonds situated at defined positions of the alkyl chains: alkenes have double bonds situated at the sixth, ninth or 12th positions, and dienes have double bonds situated at the sixth and ninth, or ninth and twelfth positions. Finding a homologous series of unsaturated aldehydes and fatty acids suggests that these alkenes and dienes are biosynthesized by a series of parallel pathways of fatty-acid elongation and desaturation reactions, which are followed by sequential reduction and decarbonylation. In addition, the silk cuticular waxes contain metabolically related unsaturated long-chain methylketones, which probably arise via a decarboxylation mechanism. Finally, metabolite profiling analyses of the cuticular waxes of two maize inbred lines (B73 and Mo17), and their genetic hybrids, have provided insights into the genetic control network of these biosynthetic pathways, and that the genetic regulation of these pathways display best-parent heterotic effects.     

Effects of host plant environment and Ustilago maydis infection on the fungal endophyte community of maize (Zea mays)

The focus of many fungal endophyte studies has been how plants benefit from endophyte infection. Few studies have investigated the role of the host plant as an environment in shaping endophyte community diversity and composition. The effects that different attributes of the host plant, that is, host genetic variation, host variation in resistance to the fungal pathogen Ustilago maydis and U. maydis infection, have on the fungal endophyte communities in maize (Zea mays) was examined. The internal transcribed spacer (ITS) region of the rDNA was sequenced to identify fungi and the endophyte communities were compared in six maize lines that varied in their resistance to U. maydis. It was found that host genetic variation, as determined by maize line, had significant effects on species richness, while the interactions between line and U. maydis infection and line and field plot had significant effects on endophyte community composition. However, the effects of maize line were not dependent on whether lines were resistant or susceptible to U. maydis. Almost 3000 clones obtained from 58 plants were sequenced to characterize the maize endophyte community. These results suggest that the endophyte community is shaped by complex interactions and factors, such as inoculum pool and microclimate, may be important.     

Water stress‐induced abscisic acid accumulation in relation to reducing agents and sulfhydryl modifiers in maize plant

Signalling process of water stress‐induced abscisic acid (ABA) accumulation was investigated in maize (Zea mays L.) leaf and root tissues. Potent free‐radical scavengers and reducing agents, N‐acetyl cysteine (NAC) and cystein (Cys), significantly inhibited or nearly completely blocked dehydration‐induced ABA accumulation. Dithiothreitol (DTT), a reducing agent but not a free‐radical scavenger, also significantly inhibited such accumulation whereas solely free‐radical scavengers, dimethyl sulphoxide (DMSO) and melatonin (Mela), had no effects. Moreover, water stress‐induced ABA accumulation was not affected either by free radicals, such as superoxide anion and hydrogen peroxide, or by oxidants such as KIO_4. These observations suggest that the blocking of water stress‐induced ABA accumulation resulted from the reducing effect, rather than from anything associated with free radicals. The disulphide bond might be crucial to the reactivity of some signal element(s) in the signalling process of water stress‐induced ABA accumulation. To test the hypothesis, we used a sulfhydryl modifier, iodoacetamide (IOA), and found that it nearly totally blocked the water stress‐induced ABA accumulation. Furthermore, an impermeable sulfhydryl modifier, p‐chloromercuriphenylsulphonic acid (PCMBS), could also inhibit the water stress‐induced ABA accumulation in the leaf tissues. These results indicate that water stress‐perception protein(s) or receptor(s) may be located on the plasmalemma and a sulfhydryl group in the extracellular domain is critical to the reactivity of the speculated water stress receptors. Cys, DTT and IOA did not lead to a decrease of the baseline ABA level, i.e. in non‐stressed roots. Result indicates that their blocking of water stress‐induced ABA accumulation occurred upstream of the ABA biosynthesis pathway, i.e. in the signalling process that initiates such accumulation.     

Biopriming of maize seeds with plant growth-promoting bacteria isolated from the earthworm Aporrectodea molleri: effect on seed germination and seedling growth

Earthworms have become a potential source of multi-beneficial bacteria and effective bioinoculants. Seed biopriming is an efficient inoculation method to apply bacteria prior to sowing, which enhances the chances of bacterial candidates to colonize the rhizosphere and/or establish a liaison with the plant. In this study, we evaluated plant growth-promoting traits of bacterial strains isolated from the earthworm’s Aporrectodea molleri chloragogenous tissue. In addition, we investigated their prospective use as biopriming agents to enhance Zea mays germination and seedling growth. Results were subjected to principal component analysis for potential correlations between the studied parameters. The bacterial strains displayed different in vitro plant growth-promoting characteristics and were efficient when applied in vivo as they significantly increased maize germination rate (26–78%), root elongation (67–84%), seedlings fresh weight and dry weight. Aeromonas encheleia TC22 was the most significant strain to influence germination due to its high ability to produce indole-3-acetic acid, and along with Pseudomonas azotoformans TC1, they were the most proficient at enhancing seedling root elongation and biomass, which was significantly correlated with their in vitro plant growth-promoting traits. Our findings indicate that isolates TC22 and TC1 are potent bio-primers for maize seeds and should be tested further for their use as biopriming inoculants.     

Similar attractiveness of maize volatiles induced by Helicoverpa armigera and Pseudaletia separata to the generalist parasitoid Campoletis chlorideae

Campoletis chlorideae Uchida (Hymenoptera: Ichneumonidae), a major larval endoparasitoid of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), also attacks many other noctuid caterpillars. We investigated the attractiveness of H. armigera‐ and Pseudaletia separata (Lepidoptera: Noctuidae)‐infested maize [Zea mays L. (Poaceae)] plants to C. chlorideae, and analyzed the volatiles emitted from infested plants and undamaged plants. Considering the reported specific induction of plant volatiles by elicitors in the caterpillar regurgitant, we also tested the response of the parasitoid to mechanically damaged plants treated with caterpillar regurgitant or water and measured the volatiles released by these plants. In wind‐tunnel bioassays, C. chlorideae was strongly attracted to herbivore‐induced maize volatiles. Mechanically damaged plants, whether they were treated with caterpillar regurgitant or water, were more attractive to the parasitoid than undamaged plants. The parasitoid did not distinguish between maize seedlings infested by the two noctuid insects, nor did they show a difference in attraction to mechanically damaged plants treated with caterpillar regurgitant or water. Coupled gas chromatography–mass spectrometer (GC‐MS) analysis revealed that 15 compounds were commonly emitted by herbivore‐infested and mechanically damaged maize plants, whereas only two compounds were released in minor amounts from undamaged plants. Infestation by H. armigera specifically induced four terpenoids, β‐pinene, β‐myrcene, D‐limonene, and (E)‐nerolidol, which were not induced by infestation of P. separata and mechanical damage, plus caterpillar regurgitant or water. Two compounds, geranyl acetate and β‐sesquiphellandrene, were also induced by the infestation of H. armigera, but not by the infestation of P. separata. All treated maize plants released volatiles in significantly larger total amounts than did undamaged plants. Maize plants infested by H. armigera emitted greater amounts of volatiles than plants infested by P. separata. The treatment with caterpillar regurgitant resulted in larger amounts of volatile emission than the treatment with water did in mechanically damaged plants. The amounts of emissions of individual compounds were also different between differently treated plants.     

Proteomic analysis of the maize rachis: potential roles of constitutive and induced proteins in resistance to Aspergillus flavus infection and aflatoxin accumulation

Infection of the maize (Zea mays L.) with aflatoxigenic fungus Aspergillus flavus and consequent contamination with carcinogenic aflatoxin is a persistent and serious agricultural problem causing disease and significant crop losses worldwide. The rachis (cob) is an important structure of maize ear that delivers essential nutrients to the developing kernels and A. flavus spreads through the rachis to infect kernels within the ear. Therefore, rachis plays an important role in fungal proliferation and subsequent kernel contamination. We used proteomic approaches and investigated the rachis tissue from aflatoxin accumulation resistant (Mp313E and Mp420) and susceptible (B73 and SC212m) maize inbred lines. First, we compared rachis proteins from resistant and susceptible inbred lines, which revealed that the young resistant rachis contains higher levels of abiotic stress-related proteins and proteins from phenylpropanoid metabolism, whereas susceptible young rachis contains pathogenesis-related proteins, which are generally inducible upon biotic stress. Second, we identified A. flavus-responsive proteins in rachis of both resistant and susceptible genotypes after 10- and 35-day infection. Differential expression of many stress/defense proteins during rachis juvenility, maturation and after A. flavus challenge demonstrates that resistant rachis relies on constitutive defenses, while susceptible rachis is more dependent on inducible defenses.     

Immunological evidence for the presence of plant homologues of the actin- related protein Arp3 in tobacco and maize: subcellular localization to actin-enriched pit fields and emerging root hairs

Summary. The actin-nucleating and -organizing Arp2/3 protein complex is well known to be conserved throughout the eukaryotic kingdom. For higher plants, however, only limited evidence is available for the presence of the Arp2/3 complex so far. Using heterologous antibodies against the Dictyostelium discoideum and Schizosaccharomyces pombe proteins and a bovine peptide, we found immunological evidence for the presence of Arp3 homologues in plants. First, proteins with a molecular mass of about 47–50 kDa were clearly recognized in extracts of both a dicotyledonous plant (tobacco) and a monocotyledonous plant (maize) in immunoblots with the anti-Arp3 antibodies. Second, immunolocalization with these Arp3 antibodies was performed on different plant cells, selected for their diverse actin organizations and functions. On isolated plasma membrane ghosts derived from tobacco leaf protoplasts, a putative Arp3 was localized along cortical actin filaments. In the inner cortex of maize roots, Arp3 was localized to actin-rich plasmodesmata and pit fields and to multivesicular bodies in the cytoplasm. During root hair formation, distinct site-specific localization was found at the protruding apical plasma membrane portions of these tip-growing cells.Correspondence and reprints: Department of Biology, Universitaire Instelling Antwerpen, Universiteitsplein 1, 2610 Wilrijk, Belgium.Received January 3, 2003; accepted February 7, 2003; published online August 26, 2003     

Drought-induced changes in anthesis-silking interval are related to silk expansion: a spatio-temporal growth analysis in maize plants subjected to soil water deficit

The growth and emergence of maize silks has a considerable importance in yield determination under drought conditions. Spatial and temporal patterns of the rates of tissue expansion and of cell division were characterized in silks of plants subjected to different soil water potentials. In all cases, silk development consisted of four phases: (1) cell division and tissue expansion occurred together uniformly all along the silk; (2) cell division progressively ceased from tip to base, while expansion remained spatially uniform including during the phase (3) after the cessation of cell division; and (4) as the silk emerged from the husks, expansion ceased in the emerged portion, probably because of direct evaporative demand, while the relative growth rate progressively decreased in the enclosed part. The rates of tissue expansion and cell division were reduced with water deficit, resulting in delayed silk emergence. The duration of cell division was not affected, and in all cases, the end of cell division in the silk coincided with anther dehiscence. The duration of phase 3, between the end of cell division and the arrest of cell growth in silk apex, considerably increased with water deficit. It corresponded to the anthesis-silking interval used by breeders to characterize the response of cultivars to stress.     

Targeted transcriptomic and metabolic profiling reveals temporal bottlenecks in the maize carotenoid pathway that may be addressed by multigene engineering

Carotenoids are a diverse group of tetraterpenoid pigments found in plants, fungi, bacteria and some animals. They play vital roles in plants and provide important health benefits to mammals, including humans. We previously reported the creation of a diverse population of transgenic maize plants expressing various carotenogenic gene combinations and exhibiting distinct metabolic phenotypes. Here we performed an in‐depth targeted mRNA and metabolomic analysis of the pathway to characterize the specific impact of five carotenogenic transgenes and their interactions with 12 endogenous genes in four transgenic lines representing distinct genotypes and phenotypes. We reconstructed the temporal profile of the carotenoid pathway during endosperm development at the mRNA and metabolic levels (for total and individual carotenoids), and investigated the impact of transgene expression on the endogenous pathway. These studies enabled us to investigate the extent of any interactions between the introduced transgenic and native partial carotenoid pathways during maize endosperm development. Importantly, we developed a theoretical model that explains these interactions, and our results suggest genetic intervention points that may allow the maize endosperm carotenoid pathway to be engineered in a more effective and predictable manner.