Fungal genes related to calcium homeostasis and signalling are upregulated in symbiotic arbuscular mycorrhiza interactions

Fluctuations in intracellular calcium levels generate signalling events and regulate different cellular processes. Whilst the implication of Ca²⁺ in plant responses during arbuscular mycorrhiza (AM) interactions is well documented, nothing is known about the regulation or role of this secondary messenger in the fungal symbiont. The spatio-temporal expression pattern of putatively Ca²⁺-related genes of Glomus intraradices BEG141 encoding five proteins involved in membrane transport and one nuclear protein kinase, was investigated during the AM symbiosis. Expression profiles related to successful colonization of host roots were observed in interactions of G. intraradices with roots of wild-type Medicago truncatula (line J5) compared to the mycorrhiza-defective mutant dmi3/Mtsym13. Symbiotic fungal activity was monitored using stearoyl-CoA desaturase and phosphate transporter genes. Laser microdissection based-mapping of fungal gene expression in mycorrhizal root tissues indicated that the Ca²⁺-related genes were differentially upregulated in arbuscules and/or in intercellular hyphae. The spatio-temporal variations in gene expression suggest that the encoded proteins may have different functions in fungal development or function during symbiosis development. Full-length cDNA obtained for two genes with interesting expression profiles confirmed a close similarity with an endoplasmic reticulum P-type ATPase and a Vcx1-like vacuolar Ca²⁺ ion transporter functionally characterized in other fungi and involved in the regulation of cell calcium pools. Possible mechanisms are discussed in which Ca²⁺-related proteins G. intraradices BEG141 may play a role in mobilization and perception of the intracellular messenger by the AM fungus during symbiotic interactions with host roots.     

Mycorrhizas: Gene to Function

Substantial progress has been made toward development of molecular tools for identification and quantification of mycorrhizal fungi in roots and evaluation of the diversity of ectomycorrhizal (ECM) fungi and the phylogeny and genetic structure of arbuscular mycorrhizal (AM) fungi. rDNA analysis confirms high diversity of ECM fungi on their hosts, and for AM fungi has revealed considerable genetic variation within and among morphologically similar AM fungal species. The fungal and plant genes, regulation of their expression, and biochemical pathways for nutrient exchange between symbiotic partners are now coming under intense study and will eventually be used to define the ecological nutritional role of the fungi. While molecular biological approaches have increased understanding of the mycorrhizal symbiosis, such knowledge about these lower-scale processes has yet to influence our understanding of larger-scale responses to any great extent.     

Plant signals and fungal perception during arbuscular mycorrhiza establishment

Arbuscular mycorrhizal (AM) fungi are obligate symbionts that need their plant hosts to complete their life cycle. In the absence of the plant, germlings arrest growth after a few days and retract most of their cytoplasm back into the multinuclear spores. The spores can germinate again during more favorable conditions. How AM fungi recognize compatible host roots and activate their symbiotic program is not yet understood. However, research in this field in the last years has shed light into this topic. We, and others, have approached some of these aspects by studying changes in fungal gene expression observed at early stages of development, before and at the plant recognition stage in an attempt to identify genes and proteins featuring as key regulators in the switch between the asymbiotic and symbiotic style of life. The molecular bases of this recognition process are now starting to be understood and point to common signaling pathways shared with other microbe-plant associations and to arbuscular mycorrhiza specific signaling pathways.     

Aquaporin genes GintAQPF1 and GintAQPF2 from Glomus intraradices contribute to plant drought tolerance

Arbuscular mycorrhizal (AM) symbiosis, established between AM fungi (AMF) and roots of higher plants, occurs in most terrestrial ecosystems. It has been well demonstrated that AM symbiosis can improve plant performance under various environmental stresses, including drought stress. However, the molecular basis for the direct involvement of AMF in plant drought tolerance has not yet been established. Most recently, we cloned two functional aquaporin genes, GintAQPF1 and GintAQPF2, from AM fungus Glomus intraradices. By heterologous gene expression in yeast, aquaporin localization, activities and water permeability were examined. Gene expressions during symbiosis in expose to drought stress were also analyzed. Our data strongly supported potential water transport via AMF to host plants. As a complement, here we adopted the monoxenic culture system for AMF, in which carrot roots transformed by Ri-T DNA were cultured with Glomus intraradices in two-compartment Petri dishes, to verify the aquaporin gene functions in assisting AMF survival under polyethylene glycol (PEG) treatment. Our results showed that 25% PEG significantly upregulated the expression of two aquaporin genes, which was in line with the gene functions examined in yeast. We therefore concluded that the aquaporins function similarly in AMF as in yeast subjected to osmotic stress. The study provided further evidence to the direct involvement of AMF in improving plant water relations under drought stresses.     

Expression studies of plant genes differentially expressed in leaf and root tissues of tomato colonised by the arbuscular mycorrhizal fungus Glomus mosseae

Arbuscular mycorrhizal (AM) fungi are a multifaceted group of mutualistic symbionts that are common to terrestrial ecosystems. The interaction between AM fungi and plant roots is of environmental and agronomic importance. Understanding the molecular changes within the host plant upon AM fungal colonisation is a pre-requisite to a greater understanding of the mechanisms underlying the interaction. Differential mRNA display was conducted on leaf tissue of tomato plants colonised and non-colonised by the AM fungus Glomus mosseae and five putative differentially regulated cDNAs were identified. All cDNAs isolated shared high sequence similarity to known plant genes. Differential screening was initially used to establish whether the cDNAs were differentially expressed. Semi-quantitative RT-PCR was used to establish gene expression patterns for all five clones within leaf and root tissue of mycorrhizal and non-mycorrhizal colonised tomato plants. Differential regulation was observed for all five cDNAs. Down-regulation within the leaf tissue of mycorrhizal plants was observed for 4 out of the 5 cDNAs with an up-regulation observed only for one. Tissue specific regulation was observed for several cDNAs, with down-regulation observed in mycorrhizal leaf tissue and up-regulation observed within mycorrhizal root tissue as compared to non-mycorrhizal tissue.     

Gibberellin regulates infection and colonization of host roots by arbuscular mycorrhizal fungi

Arbuscular mycorrhiza (AM) is established by the entry of AM fungi into the host plant roots and the formation of symbiotic structures called arbuscules. The host plant supplies photosynthetic products to the AM fungi, which in return provide phosphate and other minerals to the host through the arbuscules. Both partners gain great advantages from this symbiotic interaction, and both regulate AM development. Our recent work revealed that gibberellic acids (GAs) are required for AM development in the legume Lotus japonicus. GA signaling interact with symbiosis signaling pathways, directing AM fungal colonization in host roots. Expression analysis showed that genes for GA biosynthesis and metabolism were induced in host roots around AM fungal hyphae, suggesting that the GA signaling changes with both location and time during AM development. The fluctuating GA concentrations sometimes positively and sometimes negatively affect the expression of AM-induced genes that regulate AM fungal infection and colonization.     

Is stimulation of carotenoid biosynthesis in arbuscular mycorrhizal roots a general phenomenon?

The identification and quantification of cyclohexenone glycoside derivatives from the model legume Lotus japonicus revealed far higher levels than expected according to the stoichiometric relation to another, already determined carotenoid cleavage product, i.e., mycorradicin. Mycorradicin is responsible for the yellow coloration of many arbuscular mycorrhizal (AM) roots and is usually esterified in a complex way to other compounds. After liberation from such complexes it has been detected in AM roots of many, but not of all plants examined. The non-stoichiometric occurrence of this compound compared with other carotenoid cleavage products suggested that carotenoid biosynthesis might be activated upon mycorrhization even in plant species without detectable levels of mycorradicin. This assumption has been supported by inhibition of a key enzyme of carotenoid biosynthesis (phytoene desaturase) and quantification of the accumulating enzymic substrate (phytoene). Our observations suggest that the activation of carotenoid biosynthesis in AM roots is a general phenomenon and that quantification of mycorradicin is not always a good indicator for this activation.     

The screening and selection of inoculant arbuscular-mycorrhizal and ectomycorrhizal fungi

The initiation of a programme of screening and selection of arbuscular-mycorrhizal fungi (AM fungi) and ectomycorrhizal fungi (ECM fungi) for use as inoculant fungi in agriculture, horticulture of forestry will depend on whether inoculation is more appropriate as a management option than manipulation of the indigenous mycorrhizal populations. The greatest immediate potential for the successful use of mycorrhizal inoculants will be in soils and growth media where phosphorus (P) limits plant growth and where the indigenous mycorrhizal fungi are either ineffective at increasing P uptake by the plant or their numbers have been drastically reduced by human influence or natural disturbance. In recent investigations, however, additional benefits to the plant following colonization of roots by effective AM or ECM fungi have been demonstrated. These additional benefits of an effective mycorrhizal association have necessitated a re-evaluation of the optimum screening procedures for isolates of AM and ECM fungi. Both current methodologies and suggestions for alternative approaches to the screening of AM and ECM fungi are discussed in this paper. The problems inherent in choosing suitable experimental conditions to compare different isolates at the screening stage are also addressed. The review also stresses the importance of continued monitoring of introduced mycorrhizal fungi to learn more about their longer-term ecological role, particularly within reforestation or revegetation studies.     

Do Sebacinales commonly associate with plant roots as endophytes?

Sebacinales are basal Hymenomycetes with diverse mycorrhizal abilities, ranging from ectomycorrhizae to ericoid and orchid mycorrhizae. Several previous PCR or isolation works raised the possibility that Sebacinales are endophytes in plant roots. We tested this hypothesis in an isolation-independent approach by using specific PCR primers for ribosomal DNA of Sebacinales on AM mycorrhizal or non-mycorrhizal roots. Thirty-nine plant species were sampled on a Caribbean and two European sites (3 repetition per species and site), covering 25 families in monocots and eudicots. PCR signals were obtained from 40 samples (28.9 %) from 27 species (69.2 %) and all sites. Whenever sequencing was successful, a sequence belonging to Sebacinales was recovered. A phylogenetic approach revealed that 13 of them belonged to clade B (encompassing ericoid and orchid mycorrhizal species) and 4 to clade A (usually encompassing only ectomycorrhizal species). These data suggest that Sebacinales may be endophytic in many angiosperm roots, and that this condition is plesiomorphic in Sebacinales. They bridge the gap between physiological studies, inoculating Sebacinales (Piriformospora indica or Sebacina vermifera) on diverse plants and molecular ecology, hitherto restricting Sebacinales to mycorrhizal interactions. Structural and functional aspects of the interaction deserve further studies.     

亚热带典型树种根毛特征及其与共生真菌的关系

根毛和共生真菌增加了吸收面积,提高了植物获取磷等土壤资源的能力。由于野外原位观测根表微观结构较为困难,吸收细根、根毛、共生真菌如何相互作用并适应土壤资源供应,缺乏相应的数据和理论。该研究以受磷限制的亚热带森林为对象,选取了21种典型树种,定量了根毛存在情况、属性变异,分析了根毛形态特征与共生真菌侵染率、吸收细根功能属性之间的关系,探讨了根表结构对低磷土壤的响应和适应格局。结果表明:1)在亚热带森林根毛不是普遍存在的, 21个树种中仅发现7个树种存有根毛, 4个为丛枝菌根(AM)树种, 3个为外生菌根(ECM)树种。其中,马尾松(Pinus massoniana)根毛出现率最高,为86%;2)菌根类型是理解根-根毛-共生真菌关系的关键,AM树种根毛密度与共生真菌侵染率正相关,但ECM树种根毛直径与共生真菌侵染率负相关; 3) AM树种根毛长度和根毛直径、ECM树种根毛出现率与土壤有效磷含量呈负相关关系。该研究揭示了不同菌根类型树种根毛-共生真菌-根属性的格局及相互作用,为精细理解养分获取策略奠定了基础。