A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae

Abstract

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1×10 6 ml −1 , the percentages of conidium germination and appressorium formation were (97.98±0.67)% and (97.88±0.45)%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 μg total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA (complementary DNA) library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.

MoRad6‐mediated ubiquitination pathways are essential for development and pathogenicity inMagnaporthe oryzae

“…To analyze gene expression levels, RNA samples were isolated using RNAiso kit (Takara, Japan) following the manufacturer's instructions from the wild‐type strain during four developmental stages, including mycelia cultured in CM for 3 days, conidia collected from 10‐day‐old CM plates, appressoria induced on hydrophobic surface 18 hpi, infected rice leaves harvested 4 dpi, and mycelia of the generated mutants (Liu et al ., ). The cDNA was synthesized with reverse transcript kit (Takara, Japan).…”

The cysteine protease MoAtg4 interacts with MoAtg8 and is required for differentiation and pathogenesis inMagnaporthe oryzae

“…25,26 First-strand cDNA was generated using M-MLV Reverse Transcriptase (Promega, M1701). A pair of primers (named ATG4 RTF and ATG4 RTR, see Table S1) was designed for MoATG4 stage expression pattern analysis.…”

Involvement of MoVMA11, a Putative Vacuolar ATPase c’ Subunit, in Vacuolar Acidification and Infection-Related Morphogenesis of Magnaporthe oryzae

“…Total RNAs of the above samples were isolated with the Trizol reagent (Takara) following a previously described protocol [33]. After the synthesis of first strand cDNA from 800 ng of total RNA using SYBR ExScriptTM RT-PCR kit (Takara), real-time PCR reaction was performed with SYBR Premix Ex Taq (Takara) on a Mastercycler ep realplex thermo cycler (Eppendorf) [34].…”