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Genome-wide functional analysis reveals that infection-associated fungal…
Genome-wide functional analysis reveals that infection-associated fungal autophagy is necessary for rice blast disease
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Discussion
:star: Autophagy is a cell survival response that is triggered normally by starvation stress & used to recycle cytoplasm, organelle and proteins within cells.
:star: Autophagy important in heterokaryon incompatibility in Podospora anserina & for metal ion homeostasis in Aspergillus fumigatus
:star: Autophagy necessary for cellular differentiation, defense from infection & many aspects of development in multicellular organism.
In previous study:
:black_circle: M. oryzae demonstrated a role for MoATG8
in autophagic cell death of conidia during appressorium development.
:black_circle: Moatg8 mutants were unable to undergo conidial cell collapse, and although they could form appressoria, these were non-functional and unable to cause plant disease.
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:star: Autophagy has also been shown to be a contributing factor in cell death, indicating that a non-apoptotic programmed cell death pathway exists in eukaryotes that is dependant on autophagy genes.
:star: Induction of autophagy during infection-related development is developmentally regulated and requires the Pmk1 MAP kinase pathway, but is also consequence of starvation stress.
:check: Purpose of autophagy: to fuel infection-related development in the absence of exogenous nutrients before entry into the plant.
For this reason, it has to be tightly couple to genetic control of development of the appressorium, which occurs via the cell cycle since completion of mitosis is a necessary prerequisite both for appressorium maturation and conidial death.
The absence of any component of the autophagic machiney is sufficient to prevent both conidial collapse and appressorium-mediated plant infection.
:star: Major aim in this study was to test whether infection-associated autophagy is a selective or non-selective process, which could not determined by analysis of MoATG8.
Introduction
To determine whether:
- infection-related
autophagy is necessary for rice blast disease solely as a result of its role in conidial cell death
- or whether appressoria also undergo autophagy during their maturation
- define whether autophagy carried out by M.oryzae during plant infection is a selective or non-selective autophagy
- Yeast S. cerevisiae has identified a family of 30 ATG genes, which encode proteins for autophagy
- TOP kinase regulates initiation of autophagy
- the autophagosomes expands & fuses with a vacuole
- adopt a genomic-wide approach: systematically analyze the autophagic machinery of M.oryzae (to determine why fungal autophagy is necessary for rice blast)
first developed a rapid method for gene functional analysis in M.oryzae and deployed this method to characterize the 22 fungal genes involved in autophagy
Conclusion: infection-related autophagy is nonselective and takes place in both conidia and appresoria of M.oryzae leading to death of death of the conidium and development of afunctional appressorium essential for plant disease.
Methods
*Generation of the ku70 Mutant of M. oryzae*.**
- the MoKu70 gene identified and primers designed to amplify the 2 regions flanking the gene.
- primers used : Ku70F1 & Ku70LFr (to amplify 1.0 kb region upstream - from start codon; introduce a NdeI site at the 3' end and primers Ku7-RFf & Ku70RFr to amplify a 1.0 kb region downstream.
- 2 flanking DNA fragments cloned into pGEM-T & the left flank excised with NdeI/NotI and cloned into the vector with the right flank, giving pMG12.1
- 2.8 kb fragment using
primers SurF and SurR introducing an NdeI site to both ends of the amplicon (create pMJG2)
- Notl & Apal restriction sites within the pGEM-T polylinker used to liberate the gene disrupt cassette from pMLG2
Generation of Moatg Mutants
- targated gene replacement used split marker strategy.
- vector is constructed using a hygromycinB resistance selectable marker, hph, for transformation of Moku70.
- The hph gene cassette was cloned into pBluescript as a 1.4 kb EcoRI-XbaI fragment.
- M13 with HY & M13R with YG used to amplify the spilt hph template.
GFP:MoATG8 Gene Fusion Construction.
- MoATG8 amplified as a 1.6 kb fragment using primers ATG8.5 & ATG8.3
- The amplicon was digested & cloned into pCB1532
- The promoter region of the MoATG8 gene was amplified as a 1.4 kb fragment using primers ATG8p5 and Atg8p3
- The fragment was sub cloned into pMJK142.2 in frame with sGFP gene as a SpeI - NcoI fragment
Light and Epifluorescence Microscopy.
- Epifluorescence microscopy to visualize
eGFP and MDC-stained samples
- epifluorescence examination of the GFP:MoATG8 transformants, conidia were incubated onto coverslips and placed onto a 2% agar
