Hydroxyproline-rich glycoprotein mRNA accumulation in maize root cells colonized by an arbuscular mycorrhizal fungus as revealed by in situ hybridization

Summary To determine whether the expression of cell wall related genes changes during the establishment of an arbuscular mycorrhizal symbiosis (AM), we studied the expression of a maize hydroxyproline-rich glycoprotein (HRGP) gene. In situ hybridization showed that, in differentiated cells of maize roots, mRNA accumulation corresponding to the gene encoding for HRGP was only found when the cells were colonized by the endomycorrhizal fungusGlomus versiforme.     

Location of a cell-wall hydroxyproline-rich glycoprotein,cellulose and β-1,3-glucans in apical and differentiated regions of maize mycorrhizal roots

The cell-wall components of the interface compartment in functioning mycorrhizal roots of maize (Zea mays L. cv. W64A) have been investigated with the use of immunocytochemistry and enzyme/lectin-gold techniques. The distribution of specific cell-wall probes was determined in the apical and differentiated regions of maize roots in the presence and in the absence of the mycorrhizal fungus, Glomus versiforme. Labelling experiments showed that a maize hydroxyproline-rich glycoprotein (HRGP), identified with a specific antibody, was particularly abundant in the apical dividing cells of the root meristem. Cellulose, located with a cellobiohydrolase-gold complex, showed a similar labelling pattern in the walls of both meristematic and differentiated parts of the roots. When the cortex was colonized by the mycorrhizal fungus, the HRGP and cellulose were expressed in two sites: the wall and the interface area created by invagination of the host membrane around the developing fungus. In contrast, in uninfected roots of the same age, they were only present in the inner part of the wall. A specific antibody against -1,3-glucans demonstrated that these glucans were not laid down at the interface between the plant and fungus, while they appeared to be a skeletal component of the fungal wall, together with chitin.Abbreviations CBH I cellobiohydrolase - DAPI 4,6-diamino-2-phenylindole - HRGP hydroxyproline-rich glycoprotein The research was supported by the Italian Murst (40%) grant and by an International Project between Spain and Italy (Azioni Integrate).     

Localization of the yellow pigment formed in roots of gramineous plants colonized by arbuscular fungi

Summary Many plants form yellow coloured roots when colonized by arbuscular mycorrhizal (AM) fungi. In maize, a yellow pigment is first visible as small droplets in parenchyma cells of roots in the vicinity of arbuscules, 3–4 weeks after mycorrhizal colonization. During the course of the development of the plants, the yellow pigment spreads all over the cells of the cortex (with the exception of the exodermis) and of the endodermis, whereas the other stelar elements remain uncoloured. Other gramineous plants (wheat, barley, millet) show the same pattern of pigment formation. In contrast, the deposition of this pigment is not detected in roots ofTagetes, garden bean, onion, or leek. Weak yellow fluorescence is also seen in the fungal structures, particularly in the arbuscules of the investigated probes. This is, however, clearly different from the intense yellow colour of the pigment formed in root cells of grasses. The yellow pigment is even detected in such cells which are never colonized by fungal structures (e.g., endodermal cells). A major constituent of the yellow pigment of AM-colonized root cells has been identified as a carotenoid with 14 carbon atoms and two carboxylic groups and termed mycorradicin. This carotenoid is likely deposited in the vacuoles of root cells as a result of the colonization specifically by arbuscular fungi.     

Chib1和PAL5基因在AM真菌Glomus fasciculatus诱导大豆抗线虫病害防御反应中的作用

本研究系统分析了大豆（品种：‘鲁豆4’）接种AM真菌Glomus fasciculatum和胞囊线虫（SCN,Heterodera glycines）4号生理小种后各处理菌根和线虫侵染率、几丁质酶和苯丙氨酸解氨酶（PAL）活性及几丁质酶基因Chib1和苯丙氨酸解氨酶基因PAL5转录物的动态变化。结果表明,接种SCN对AM真菌的侵染率没有产生显著影响,但先接种AM真菌后接种SCN的大豆根内线虫侵染率明显低于只接种SCN的处理。另外,先接种AM真菌后接种SCN的大豆根内几丁质酶和PAL活性显著提高,活性高峰出现在接种线虫后的第3天。值得注意的是,先接种AM真菌后接种SCN的大豆根内两种基因Chib1和PAL5转录物高峰也出现在接种SCN后的第3天,即AM真菌侵染率快速上升而SCN侵染率快速下降时期。所以Chib1和PAL5基因的表达可能是AM真菌诱导的抗大豆胞囊线虫病害防御反应的一种表现。因此推测Chib1和PAL5直接参与了AM真菌诱导大豆抗胞囊线虫病害的防御反应。     

Spatial distribution of chitinases and β-1,3-glucanase transcripts in bean arbuscular mycorrhizal roots under low and high soil phosphate conditions

Arbuscular mycorrhizal (AM) fungi extensively colonize the root cortex under low-soil-phosphate (P) conditions, whereas infection is limited under high-P conditions. Fungal growth under both P conditions might be influenced by plant defence-related gene expression. In this study, we used in situ hybridization methods to compare the cellular localization of three defence-related mRNAs in non-infected bean roots and in relation to fungal infection units. In non-infected and infected roots, mRNAs encoding acidic and basic endochitinases were generally most abundant in the vascular cylinder. High-P-grown mycorrhizal roots showed localized accumulation of the acidic endochitinase mRNA in cortical cells containing arbuscules and in their immediate vicinity (one to five cell layers). The pattern of accumulation of the basic endochitinase mRNA was not affected by P or AM fungal infection. At the low P concentration, the β-1,3-glucanase mRNA accumulated predominantly in the vascular cylinder of non-infected roots. Suppression of β-1,3-glucanase mRNA accumulation in these tissues was observed in non-infected roots at the high-P and in mycorrhizal roots at both P concentrations. The observed suppression extends at least several mm from fungal infection units, characterizing a systemic effect. Beta-1,3-glucanase mRNA accumulated also around a number of cortical cells containing arbuscules only at the low P concentration. The localized accumulations of the endochitinase and β-1,3-glucanase mRNAs suggest that the encoded proteins might be involved in the control of intraradical fungal growth.     

The Medicago truncatula sucrose synthase gene MtSucS1 is activated both in the infected region of root nodules and in the cortex of roots colonized by arbuscular mycorrhizal fungi

The MtSucS1 gene encodes a sucrose synthase (EC 2.4.1.13) in the model legume Medicago truncatula. To determine the expression pattern of this gene in different organs and in particular during root endosymbioses, we transformed M. truncatula with specific regions of MtSucS1 fused to the gusAint reporter gene. These fusions directed an induction to the vasculature of leaves, stems, and roots as well as to flowers, developing seeds, young pods, and germinating seedlings. In root nodules, strong promoter activity occurred in the infected cells of the nitrogen-fixing zone but was additionally observed in the meristematic region, the prefixing zone, and the inner cortex, including the vasculature. Concerning endomycorrhizal roots, the MtSucS1 promoter mediated strongest expression in cortical cells harboring arbuscules. Specifically in highly colonized root sections, GUS-staining was furthermore detected in the surrounding cortical cells, irrespective of a direct contact with fungal structures. In accordance with the presence of an orthologous PsSus1 gene, we observed a comparable regulation of MtSucS1 expression in the grain legume Pisum sativum in response to microbial symbionts. Unlike other members of the MtSucS gene family, the presence of rhizobial or Glomus microsymbionts significantly altered and enhanced MtSucS1 gene expression, leading us to propose that MtSucS1 is involved in generating sink-strength, not only in root nodules but also in mycorrhizal roots.     

Characterization of Mycorrhizas Formed by Glomus sp. on Roots of Hypernodulating Mutants of Lotus japonicus

Lotus japonicus hypernodulating mutants, Ljsym78-1 and Ljsym78-2, by the arbuscular mycorrhizal fungus Glomus sp. was characterized. The mutants are defective in systemic autoregulation of nodulation and nitrate inhibition, and form an excess of nodules and lateral roots. The percent root length colonized by the arbuscular mycorrhizal fungi was significantly higher for the mutant than wild-type roots. Detailed assessment of the colonization indicated that the percentage of colonization by arbuscules was increased, but that by external hyphae, internal hyphae and vesicles was decreased, in the mutant roots compared with the wild-type. The succinate dehydrogenase activity of arbuscules, external hyphae and internal hyphae showed similar trends. In addition, the majority of individual arbuscules that formed on the mutant roots had a well-developed and seemingly tough morphology. The results suggest that mutation at the Ljsym78 locus positively stimulates the growth and activity of arbuscules, but leads to reduced growth and activity of hyphae. We report the first identification of Lotus japonicus mutants that show significantly increased arbuscule formation and termed these mutants Arb⁺⁺. Received 8 August 2000/ Accepted in revised form 19 October 2000     

Regulation of isoflavonoid metabolism in alfalfa

Isoflavonoids are believed to play important roles in plant-microbe interactions. During infection of alfalfa (Medicago sativa) leaves with the fungal pathogen Phoma medicaginis, rapid increases in mRNA levels and enzyme activities of isoflavone reductase, phenylalanine ammonia-lyase, chalcone synthase and other defense genes are observed within 1 to 2 hours. The phytoalexin medicarpin and its antifungal metabolite sativan increase beginning at 4 and 8 hours, respectively, along with other isoflavonoids. In contrast, during colonization of alfalfa roots by the symbiotic mycorrhizal fungus Glomus versiforme, expression of the general phenylpropanoid and flavonoid genes phenylalanine ammonia-lyase and chalcone synthase increases while mRNA levels for the phytoalexin-specific isoflavone reductase decrease. The total isoflavonoid content of colonized roots increases with time and is higher than that of uninoculated roots, but the accumulation of the antifungal medicarpin is somehow suppressed.An isoflavone reductase genomic clone has been isolated, promoter regions have been fused to the reporter gene -glucuronidase, and the promoter-reporter fusions have been transformed into tobacco and alfalfa. Using histological staining, we have studied the developmental and stress-induced expression of this phytoalexin-specific gene in whole plants at a more detailed level than other methods allow. The isoflavone reductase promoter is functional in tobacco, a plant which does not synthesize isoflavonoids. Infection of transgenic alfalfa plants by Phoma causes an increase in -glucuronidase staining, as does elicitation of transgenic alfalfa cell cultures, indicating that this promoter fusion is a good indicator of phytoalexin biosynthesis in alfalfa.Abbreviations CA4H cinnamic acid 4-hydroxylase - CHI chalcone isomerase - CHOMT chalcone O-methyltransferase - CHS chalcone synthase - 4CL 4-coumarate:CoA ligase - COMT caffeic acid O-methyltransferase - FGM malonylated glucoside of formononetin - GUS -glucuronidase - IFOH isoflavone 2-hydroxylase - IFR isoflavone reductase - IFS isoflavone synthase - IOMT isoflavone 4-O-methyltransferase - MGM medicarpin 3-O-glucoside-6-O-malonate - PAL L-phenylalanine ammonia-lyase - PTS pterocarpan synthase - VAM vesicular arbuscular mycorrhizal - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronide