GoKawiilFungal pathogen isolates and host plants
Maintenance of M. oryzae and medium composition are as described previously38. The M. oryzae wild-type strain Guy11 isolate was grown on complete medium in a growth chamber at 25 °C in a 12-h light–12-h dark photoperiod for 10 days before conidia were collected for inoculation of rice plants. Mycelial growth of M. oryzae strains in different stress conditions was measured on complete agar medium supplemented with or without chemicals for 10 days. The chemicals included 5 mM H 2 O 2 , which was supplemented to induce oxidative stress; 200 μg ml−1 congo red or calcofluor white, which was used to induce cell wall stress; and 0.01% SDS, which was used to induce cell membrane stress.
Rice infection with M. oryzae was conducted as previously described37. Conidial suspensions (1 × 105 spores ml−1) of M. oryzae strains were used to inoculate rice and incubated for 5–7 days before being photographed. The relative fungal DNA amount was calculated using the threshold cycle value (C T ) of M. oryzae POT2 DNA (MoPOT2) against C T of rice ubiquitin genomic DNA39. The infection-related development of M. oryzae was examined following previous reports40 at indicated hpi with M. oryzae strains. The frequency of plant penetration was assessed by counting the formation of appressoria and/or penetration pegs on leaf sheaths of TP309 rice japonica cultivar or its transgenic lines from at least 50 spores in triplicate on an Axio Imager A2 microscope (Zeiss). For infection-related time-course analyses, hpi denotes hours after inoculation with M. oryzae strains.
R. solani AG1 strain was grown on potato dextrose agar plates at 28 °C before mycelia were collected for inoculation according to previous reports41. The wild-type F. graminearum PH-1 strain was routinely cultured on potato dextrose agar plates at 25 °C. Construction of F. graminearum deletion mutants and infection assays with wheat seedlings and spikes were conducted following previous reports42. The rice TP309 cultivar was inoculated with M. oryzae and R. solani, whereas wheat Nanmai 660 cultivar was inoculated with F. graminearum.
DNA and RNA manipulation
DNA was extracted from samples of fungus or plant using standard cetyltrimethylammonium bromide method according to a previous report43. RNA isolation was conducted as described previously39. In brief, samples were ground into powder with liquid nitrogen and transferred to 2-ml RNase-free tubes (approximately 100 mg per tube). TRIzol (Invitrogen; 15596026CN) reagent was then used for total RNA extraction according to the manufacturer’s instructions. Total RNA of M. oryzae was extracted from samples of vegetative mycelia grown in liquid complete medium or minimal medium, conidiophore on complete medium, appressorium and invasive hypha colonizing leaves. Reverse transcription was conducted with the extracted total RNA using the HiScript III RT SuperMix for qPCR (plus gDNA wiper) kit (Vazyme; R323-01), followed by RT–qPCR amplification with the ChamQ SYBR Color qPCR Master Mix kit (Vazyme; Q411) on a NextGene QL96 instrument (Jie Lai Mei Technology). Specific procedures were conducted according to the manufacturer’s instructions.
RNA-seq analysis of M. oryzae
The M. oryzae wild-type Guy11 strain was allowed to grow under four different conditions before extraction of fungal total RNA using an RNeasy Plant Mini Kit (QIAGEN; 74904). Vegetative mycelia of M. oryzae were sampled from Guy11 grown in liquid complete medium for 48 h; conidia were sampled from Guy11 grown on complete agar medium for 10 days; the appressoria were sampled from Guy11 undergoing infection structure development on hydrophobic borosilicate glass coverslips (Thermo Fisher Scientific; 12-541-B); invasive hyphae were sampled from Guy11 that has infected rice leaf sheaths for 4 days. Two biological replicate samples were prepared for each growth condition. RNA-seq library construction and sequencing analysis were conducted as previously described39. Differentially expressed genes were identified on the basis of an adjusted P value < 0.05 using the DESeq R package44.
Generation of M. oryzae mutants
A PCR-based split-marker deletion method was used for targeted gene deletion of lnc117761 as previously described39. A 600-bp DNA region of lnc117761 was targeted for replacement by the selection marker gene HPT, which confers resistance to hygromycin B (Roche; 10843555001). For genetic complementation analysis, the lnc117761 full-length gene expression vector containing its native promoter was constructed in a plasmid containing bialaphos resistance selection marker gene BAR, using a ClonExpress II One Step Cloning Kit (Vazyme; C112). To examine the non-coding property of lnc117761, a complementation plasmid was constructed that contains an lnc117761 variant with the potential short open reading frame in lnc117761 disrupted by shift mutation using the PCR-based point mutation method. Using a similar PCR-based mutation method, another variant of lnc117761 complementation plasmid was generated to disrupt the 16 nt of lnc117761 that base-pair with rice miR5827. PCR-based point mutation was also performed to introduce single-nucleotide replacement in MBS of lnc117761 to produce different lnc117761 variants. The resulting lnc117761 constructs carrying nucleotide mutations were separately transformed into Δlnc117761 for functional complementation analysis (Supplementary Fig. 1). Supplementary Table 8 details the information for all primers, PCR templates and amplicons.
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