Construction Of Single Hyphal Analysis Platform For The Investigation Of Fungal Development

Molds spread all over the nature.Usually,they can cause mildew in food and daily necessities as well as severe diseases in plants and animals.Some of them can even produce chemical carcinogens,which may cause hepatocellular carcinoma in human.However,molds may also play essential roles in the production of some traditional foods such as cheeses,sausages and soy sauce.In order to utilize molds efficiently and safe,it is of great importance to understand the physiological processes during the growth of mold.Currenly,studies of mold physiological processes mainly rely on molecular biology approaches?Genetic engineering and genome analysis?,biochemical methods?Analysis of the mycelium extract?and microscopic techniques.Those methods usually contain time-consuming sample pretreatment procedures and cannot be used to real-time monitor living fungi.Moreover,data obtained with the methods are the mean-value from a group of fungal mycelium.Since fungus pocesses a high degree of heterogeneity in its genom and behavioral characteristics,the mean-value collected from a group of samples cannot be used to precisely describe the real physiological state of each single one.Single microbial cell analysis is critical to reveal the invisible processes such as chemical signaling between the hyphal cells,which is necessary to understand the relationship between cellular biochemistry and behavioral under population level.Therefore,in situ real-time monitoring of single living hypha is necessary for advancing the understanding of mold physiology.Reactive oxygen species?ROS?and calcium ion(Ca²⁺)are actively involved in the development and metabolism of molds.Due to lack of direct investigation methods,the intracellular dynamics of endogenous small molecules during the development of mycelia is still obsecure.Analysis of single hypha is an efficient way to study the biochemical mechanism underlying fungal development.Many single cell analysis techniques have been exploited to study bacteria and mammalian cells.Due to the unique morphology and cell structure of the molds,those techniques are not suitable for their analysis.In this project,we aim to construct a single-hypha analysis platform for investigating biological roles of small molecules in the development of Aspergillus flavus?A.flavus?.The works are summarized as follows: 1.Electrochemiluminescence-based single-hypha analysis of hydrogen peroxide released from A.flavusIn this work,the electrochemiluminescence?ECL?was applied in mold investigation for the first time.Since the presence of H₂O₂ could sensitize luminol to enhance the ECL intensity,a single-hypha analysis platform based on luminol ECL system was developed for detecting hydrogen peroxide?H₂O₂?released from single A.flavus hypha.The system shows a dynamic range of 20 to 600 nM and the detection limit of 20 nM.By incubating the fungi on the transparent electrode surface,Phorbol-12-myristate-13-acetate?PMA?and Citral?CIT?-triggered H₂O₂ secretion could be real-time monitored successfully.The single-hypha analysis platform may be a promising technique for the study of mold physiology and biochemistry,further advancing our understanding of microbial heterogeneity.2.Optical nanofiber-based single-hypha analysis of ROS in A.flavusThe ECL-based single-hypha analysis platform can be used to monitor H₂O₂ released from a single mycelium.However,the platform cannot detect intracellular ROS,in particular ROS distribution in the mycelium.In this work,we developed an optical nanofiber-based single-hypha analysis platform,which contains a nanoprobe with nanoscale temporal resolution and an electric micro-operating system.The platform can meet the requirements of precise probe position and real-time detection of intracellular molecules in a single-hypha cell.The intracellular ROS distribution in single hypha with an average diameter of ⁵ ?m was analyzed by the platform,showing the uneven distribution of ROS in single hyphal cells.The level of intracellular ROS in the apex of hypha is much higher than that in the distal segment.Even in the apex segment,intracellular ROS demonstrate a gradient distribution.The closer to the tip the location is,the higher level of intracellular ROS can be detected.Transmission electron microscopy was conducted to explore the possible reason for such a ROS distribution in fungi hypha.It is found that the distribution of intracellular ROS might have a relationship with the volume,structure and location of mitochondria in the hypha.Moreover,the intracellular distribution of ROS at the hyphal tip may be one of the essential pre-requirements for the A.flavus development.3.Roles of intracellular ROS and calcium ion in apical dominance of A.flavus during the hyphal germinationSince the septum is not formed during the hyphal germination,a newly grown hypha was divided into three parts according to the distance from the location to the apex.The intracellular ROS and Ca²⁺ levels were measured with the optical nanofiber-based single-hypha analysis platform at different time-intervals.The results show that ROS and Ca²⁺ are uniformly distributed in the cells at the beginning of spore germination.As the growth of the hyphae,the concentration gradients of ROS and Ca²⁺ are gradually established,indicating that the ROS and Ca²⁺ may be involved in the formation apical dominance of hyphae at the germination stage.The ROS scavenger?DPI?,calcium chelator?BAPTA-AM?and their mixture were introduced into the detection system,respectively.Accompany with the reduction of both ROS and Ca²⁺ levels,the growth of the drug-treated hyphae is also inhibited.Based on above results,the synergistic regulation of ROS and Ca²⁺ in the development of A.flavus during the germination stage has been proposed.In this thesis,single-hypha analysis platforms were established to investigate the release,distribution and dynamics of active small biomoleucles during the development of a typical filamental fungus,A.flavus at a single-hypha level.It is found that the release of ROS as well as the distribution of ROS and Ca²⁺ are critical to the germination and growth of hyphae.The possible role of bioactive small molecules in the development of molds has also been discussed.The implementation of this project is expected to provide a novel analytical method and a new strategy for the fundamental research of molds.