Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Bacteriology
Non Technical Summary
Lipo-chitooligosaccharides (LCOs) have been discovered about 25 years ago as the primary signals produced by rhizobia bacteria and controlling the establishment and specificity of nitrogen-fixing associations between rhizobia and legumes. More recently, these LCOs were found to be produced by arbuscular mycorrhizal fungi for the establishment of symbiotic associations with plant roots. Unexpectedly, over the last year, the Ané and Keller laboratories have discovered that most filamentous fungi, including several Aspergillus species, produce LCOs too, suggesting an ancient origin of these signaling molecules. Our preliminary data indicate that LCOs drastically affect hyphal growth and development, suggesting that these molecules could be either endogenous regulators or signals between fungi controlling their growth and development. The goal of this project is to understand the synthesis and function of LCOs in fungi using the plant pathogen, Aspergillus flavus, as a model system. This project may provide innovative means of controlling this plant pathogen for crop protection but may also, in the long term, help controlling other diseases of plants, animals, and humans.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The overall goal of this project is to understand the synthesis and function of lipo-chito-oligosaccharides (LCOs) in fungi using the plant pathogen,Aspergillus flavus, as a model system. More specifically, we intend to:Test the role of fungal chitin deacetylases in LCOsynthesis inAspergillus flavus.Explore the biological function of LCOs on fungal growth and development inAspergillus flavus.Start exploring the mechanisms of LCO perception inAspergillus flavus.
Project Methods
Objective 1: Test the role of fungal chitin deacetylases in lipo-chitooligosaccharide synthesis inAspergillus flavusExperiment 1.1: Develop mutants ofAspergillus flavusin genes encoding chitin deacetylases.Single, double, and triple mutants of the three genes encoding chitin deacetylases (XP_002374100, XP_002376539, and XP_002379279) ofAspergillus flavususing strains derived from the sequenced background NRRL 3357. Briefly, the deletion of the three genes encoding chitin deacetylase will be performed by replacing the open reading frame of each gene with anargB, pyrG,orphleoRgene in theA. flavusdouble auxotroph strain TJES20.1 derived from NRRL 3357. Around 1000 bp of the 5′-flanking and 3′-flanking regions of each ofthechitin deacetylaseopen reading frames will be amplified using two pairs of specific parental primers.Later, a gene disruption cassette containing theargB, pyrG,orphleoRgenes as a selectable marker will be constructed by a double-joint PCR strategy as described inNested primers will be designed and used for the amplification of the entire deletion cassette (5'flank-argB-3'flank), (5'flank-pyrG-3'flank), and (5'flank-phleoR-3'flank) which will be used for transformation ofA. flavusprotoplastsas described previously.The deletion of multiple chitin deacetylase genes will be performed in successive rounds of transformation. Once PCR and Southern Blot confirm the first deletion strain, it will be used as a parental strain for the deletion of the second gene, and this strain used for the deletion of the third gene.Experiment 1.2: Test the effect of mutations in genes encoding chitin deacetylases on lipo-chitooligosaccharide production inAspergillus flavus.The ability of single, double, and tripleA. flavusmutants to produce LCOs will be tested using the root hair branching assay on vetch andM. truncatula. Briefly, vetch andM. truncatulaseeds are scarified and surfaced sterilized and germinated for 24 hours on agar supplemented with 2% gibberellic acid as described in [30]. Seedlings will be grown for seven days on Fahraeus medium supplemented with 0.1 µM aminoethoxyvinylglycine (AVG) to allow root and root hair growth. AVG at low concentration is often used to stimulate the response of plants to LCOs. It also leads to shorter root hairs that are easier to observe. In parallel,A. flavusmutants will be grown in liquid minimal medium supplemented with glucose. After three days, the cultures will be filtered through a sterile cheesecloth followed by a 0.22 µm polyethersulfone membrane to remove fungal cells.After seven days, when the root hairs are mature, fungal exudates will be applied to the roots, and the plants incubated for at room temperature for 48 hours. Root hair branching will be observed using a DMi1 Leica microscope in the PD lab. Sulfated LCOs at 10-8M purified from Sinorhizobium meliloti will be used as a positive control for deformations onM. truncatula. Non-sulfated LCOs purified at 10-8M fromRhizobiumsp. IRBG74 will be used as positive controls for deformations son vetch. A solution of 0.005% ethanol will be used as a negative control since purified LCOs are stored at 10-4M in 50% ethanol.Single mutants inA. flavusgenes encoding chitin deacetylases will be tested first. If they display a weak or no decrease in LCO production, double mutants and the triple mutant will be analyzed to test their functional redundancy.Objective 2: Explore the biological function of lipo-chitooligosaccharides on fungal growth and development inAspergillus flavus.Experiment 2.1. Test the ability of various lipo-chitooligosaccharidic structures to affect the growth and development ofAspergillus flavus.Our preliminary data indicate that different LCO structures have different effects on the growth and development of a wide range of fungi, includingA. flavus. However, these data onA. flavusare preliminary and need to be confirmed. We will growA. flavusin liquid minimal medium supplemented with glucose at 250 rpm and 30oC. We will add purified sulfated or non-sulfated LCOs at 10-8M. As negative controls, we will add tetraacetyl-chitotetraose (at 10-8M as well as a mock solution of 0.005% ethanol since chitooligosaccharides and LCOs are kept at 10-4M in 50% ethanol. Biomass will be evaluated during the growth stage (24 hours post inoculation), stationary stage (48 to 72 hours post inoculation), and decline stage (96 hours post inoculation), frozen with liquid nitrogen, dried in a freeze dryer for 48 hours and weighed. The effect of LCOs, CO4, and mock solutions on germination will be conducted through a germination assay on a 96-well plate with the same conditions as mentioned in the biomass experiments.Experiment 2.2: Test the effect of mutations affecting the production of lipo-chitooligosaccharides onAspergillus flavusgrowth and development.Here, we will assess the chitin deacetylase mutants made in Experiment 1.2 for any growth impact onA. flavusincluding but not limited to biomass, germination, and aflatoxin synthesis.This work has the potential to link LCO production to fungal biology. Biomass production and germination of these chitin deacetylase mutants will be analyzed as described inExperiment 2.1and aflatoxin synthesis following routine procedures in the Co-PDs lab.Experiment 2.3: Try to rescue the effect of these mutations by exogenous addition of lipo-chitooligosaccharides.Our goal is to demonstrate that the anticipated phenotypes observed inExperiment 2.2are caused by the lack of LCOs and not due to other functions of the mutated chitin deacetylases. We will try rescuing these biomass production or germination phenotypes by the addition of purified sulfated and non-sulfated LCOs at a range of concentrations between 10-8M to 10-14M. As negative controls, we will use CO4 at the same concentrations as well as the corresponding ethanol mock solutions.Objective 3: Start exploring the mechanisms of LCO perception inAspergillus flavusWe intend to develop mutants in nine genes encoding LysM-containing proteins inA. flavusand test their effect on LCO perception(AFLA_023800, AFLA_024760, AFLA_028300, AFLA_052730, AFLA_054470, AFLA_060380, AFLA_081810, AFLA_089680, AFLA_119040). Plants and animals perceive chitin-based molecules through LysM-containing receptors. We identified seven genes inA. flavuscontaining LysM domains. We will knock-out these genes individually first as described inExperiment 1.1and test the effect on these mutations on the transcriptomic response to LCOs using RT-qPCR but also growth and development assays developed inExperiment 2.1. If necessary, multiple mutations will be stacked as described in1.1or using theself-excising selection markersix-B-rec-hygroR-sixfor making multiple gene deletions inAspergillusorusing RNAi technology established in the Co-PD's lab.