Major intrinsic proteins of Laccaria bicolor: characterization, transcript profiling and functions in ectomycorrhizal associations with Picea glauca

• Author / Creator

  Xu, Hao

• In mycorrhizal associations, water transport properties of the fungal hyphae may affect water transport of the host plants. The importance of aquaporins, water-transporting members of the Major Intrinsic Protein (MIP) family, in facilitating water transport has been widely acknowledged and extensively studied in plants. However, the structure, function, and regulation of fungal MIPs are little understood. The rapid increase in the number of sequenced fungal genomes, including Laccaria bicolor, has enabled functional and comparative genomic investigations to delineate the role that fungal MIPs play in water transport of mycorrhizal plants. In this thesis project, phylogenetic analysis of 229 fungal MIPs from 88 species has revealed that MIPs of mycorrhizal fungal species fall into four clusters delineated by functionally characterized fungal MIPs: the orthodox aquaporins, the aquaglyceroporins, the facultative fungal aquaporins, and the X intrinsic proteins (XIPs). This comparative genomics analysis, together with in silico structural characterization of predicted MIPs and recently published functional characterization of MIPS from a small number of ectomycorrhizal and arbuscular mycorrhizal species, provide new insight into MIP gene families of mycorrhizal fungi, and possible roles for fungal aquaporins in water relations of mycorrhizal plant-fungus symbioses. To further characterize mycorrhizal MIPs, in silico analyses and water transport functional assay were conducted for six MIP genes cloned from ectomycorrhizal (ECM) fungus L. bicolor strain UAMH8232 which correspond to five of seven putative MIP genes of the species, and their roles in two important mycorrhizal processes – ECM root water transport and basidiocarp formation, were investigated. The aquaporin-encoding JQ585595 from L. bicolor was selected for its high water transport capacity and high transcript abundance, and transgenic L. bicolor overexpressing JQ585595 was generated to test the role of this fungal aquaporin in facilitating water transport in ECM-associated white spruce (Picea glauca). The hypothesis was tested that root hydraulic conductivity of mycorrhizal plants would be altered by overexpression of the L. bicolor aquaporin, reflecting the increased contribution of water transport through fungal hyphae to water transport of the mycorrhizal root system. In this study, P. glauca was inoculated with wild-type (WT), mock transgenic, or L. bicolor aquaporin JQ585595-overexpressing (OE) strains and exposed to root temperatures ranging from 5°C to 20°C to examine the root water transport properties, physiological responses and the plasma membrane intrinsic protein (PIP) expression in colonized plants. Mycorrhization increased shoot water potential, transpiration, net photosynthetic rates, root hydraulic conductivity, and root cortical cell hydraulic conductivity in seedlings. At 20°C, OE plants had higher root hydraulic conductivity compared with WT plants and the increases were accompanied by higher expression of P. glauca PIP GQ03401_M18.1 in roots. Contrary to WT L. bicolor, the effects of OE fungi on root and root cortical cell hydraulic conductivities were abolished at 10°C and 5°C in the absence of major changes in the examined transcript levels of P. glauca root PIPs. The results of this study provide evidence for the importance of fungal aquaporins in root water transport of mycorrhizal plants. They also demonstrate links between hyphal water transport, root aquaporin expression and root water transport in ECM plants. Mycorrhizal fungal aquaporins can transport not only water but also small neutral molecules such as glycerol, ammonia, CO2 and NO, therefore may contribute to substrate transport and cellular signaling, which is intensively required to drive rapid cell differentiation, division and expansion in certain phases of fungal growth, such as sporocarp initiation and maturation. It was hypothesized that some of these L. bicolor MIPs involved in CO2 signaling or water transport may contribute to the primordium initiation or rapid cell expansion during basidiocarp formation. Therefore, changes in their transcript profiles during the initiation and development of the basidiocarps of L. bicolor ECM with P. glauca were investigated; and morphological changes that took place in each developmental stage were examined. Based on the previous understanding of the transport capacities of L. bicolor MIPs, the involvement of the most significantly upregulated MIPs JQ585592 and JQ585595 provided important clues concerning their possible functions during the different stages of basidiocarp development. The significant involvement of JQ585595 in ECM water transport, as well as JQ585592 and JQ585595 in basidiocarp formation, highlights that fungal MIPs are of great importance to fundamental processes taking place in ECM fungi and associated plants.

• Subjects / Keywords

  • Ectomycorrhiza-water relation
  • Root hydraulic conductivity
  • Aquaporin
  • Transmembrane facilitate transport
  • Plasma membrane intrinsic protein (PIP)
  • Basidiocarp formation
  • Mycorrhizal fungal aquaporin
  • Fungal major intrinsic protein (MIP) clusters
  • Root water transport pathways

• Graduation date

  Spring 2015

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3WP9TF7C

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.