Dual-transcriptomic datasets evaluating the effect of the necrotrophic fungus Alternaria brassicicola on Arabidopsis germinating seeds

Many fungal pathogens are carried and transmitted by seeds. These pathogens affect germination and seed quality. Their transmission from the germinating seed to seedling causes many diseases in crops. Seed defense mechanisms during germination are poorly documented. RNA-seq experiments were used to describe the molecular mechanisms involved in seed interaction with a necrotrophic fungus. Here the Arabidopsis thaliana/Alternaria brassicicola pathosystem was used to perform dual-transcriptomic approach. Arabidopsis thaliana seeds and necrotrophic fungus transcripts were identified at critical germination and seedling establishment stages. Total RNA was extracted from healthy and infected germinating seeds and seedlings at 3, 6 and 10 days after sowing. Transcript libraries were made and sequenced, then fungal and plant short reads were mapped and quantified respectively against Arabidopsis thaliana and Alternaria brassicicola reference transcriptomes. This dual-transcriptomic approach revealed that 3409, 7506 and 8589 Arabidopsis thaliana genes showed a differential expression at respectevely 3, 6 and 10 days after sowing between healthy and infected seeds, including 1192 genes differentially expressed at the three studied stages. Moreover, in this experiement, we also identified the dynamic of the transcript changes occurring at the same stages in the necrotrophic fungus concomitantly during germination and seedling establishment.


a b s t r a c t
Many fungal pathogens are carried and transmitted by seeds. These pathogens affect germination and seed quality. Their transmission from the germinating seed to seedling causes many diseases in crops. Seed defense mechanisms during germination are poorly documented. RNA-seq experiments were used to describe the molecular mechanisms involved in seed interaction with a necrotrophic fungus. Here the Arabidopsis thaliana/Alternaria brassicicola pathosystem was used to perform dual-transcriptomic approach. Arabidopsis thaliana seeds and necrotrophic fungus transcripts were identified at critical germination and seedling establishment stages. Total RNA was extracted from healthy and infected germinating seeds and seedlings at 3, 6 and 10 days after sowing. Transcript libraries were made and sequenced, then fungal and plant short reads were mapped and quantified respectively against Arabidopsis thaliana and Alternaria brassicicola reference transcriptomes. This dualtranscriptomic approach revealed that 3409, 7506 and 8589 Arabidopsis thaliana genes showed a differential expression at respectevely 3, 6 and 10 days after sowing between healthy and infected seeds, including 1192 genes differen-tially expressed at the three studied stages. Moreover, in this experiement, we also identified the dynamic of the transcript changes occurring at the same stages in the necrotrophic fungus concomitantly during germination and seedling establishment.
©  [3] and MultiQC tool [4] for mapping and quality control, DESeq2 [5] for differentially expression analysis and http://bioinformatics.psb.ugent.be/webtools/Venn/ for comparison of differential expressed genes (DEGs) in all conditions. Data format Filtered raw reads (FASTQ) Analyzed RNA-seq data files (counts and DEGs lists) Percentages of seed germination and infected seeds Description of data collection Healthy Arabidopsis thaliana seeds and A. brassicicola infected seeds were collected at three germination and post-germination time points (3, 6 and 10 days after sowing) from controlled growth chamber under a 16 h photoperiod at 22 °C/20 °C (day/night) and 70% relative humidity. RNA extracts were stored at −25 °C until sequencing. Sequence quality control was performed using FastQC [3] and MultiQC [4] . Filtered raw reads were mapped and quantified using the quasi-mapping alignment available in Salmon algorithm [2] . Fungal and plant reads were accordingly mapped to either Arabidopsis Araport 11 [6]

Value of the Data
• These data contribute to the understanding of interaction between a host plant and a necrotrophic fungus at the early stage of the plant's life cycles. This early developmental stage controlling transgenerational transmission of the fungal pathogen from seeds to the seedlings is not documented up to date. • The data benefit both plant physiologists and pathologists.
The dual-transcriptomic approach allows to describe transcriptional changes occuring concomitantly in Arabidopsis and A. brassicicola . This dataset allows the identification of candidate genes and molecular markers that reflect in one side seed defense response in Arabidopsis germinating seed and in other side virulence strategy of the necrotrofic fungus.
• This data set could be used for comparison of host/pathogen interactions at different developmental stages. Developmental kinetics at 3, 6, and 10 days after sowing, allows to describe interaction mechanisms which are specific to the germinating seed compared to those of the young seedling at the autotrophic. The response of the plant specifically induced by the infections can be characterized by a differential analysis of levels of expression between the infected and the uninfected samples.

Data Description
Plant pathogen interaction at germination and early post-germination stages need to be documented at the transcriptome level. Here is presented RNA sequencing for gene expression profiling upon A. brassicicola infection in germinating seed and at early seedling establishment using the pathosystem Arabidopsis thaliana ( Arabidopsis ) /Alternaria brassicicola ( A. brassicicola ). An optimal infection condition was determined with germination assay where seed germination and seed infection rates were scored for 10 2 , 10 3 , 10 4 , 10 5 conidia/mL inoculum concentrations, respectively ( Fig. 1 ). The optimal inoculum concentration of 10 4 conidia/mL that did not affected seed germination and produced a significant seed infection rate was selected for the experimental conditions ( Fig. 2 ) used in the RNA-seq analysis. All obtained sequence raw reads in Arabidopsis and in A. Brassicicola were deposited in the NCBI Sequence Read Archive (SRA) database under the repository name NCBI GEO with the data identification number GSRA99977 ( https: //www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE199977 ). Data were extracted from MultiQC [2] analysis ( Fig. 2 ). The total number of filtered reads obtained after sequencing and the corresponding mapping rates using Arabidopsis publicly available transcriptomes (Araport 11) [6] and A. brassicicola [7] reference transcriptomes were obtained using Salmon algorithm [3] . Count files from A. brassicicola and Arabidopsis were for all three replicates and were used to identify differentially expressed genes between healthy and infected seeds at 3, 6 and 10 days after sowing. The pair-wise comparisons between healthy and infected host plant transcripts according to DE-seq2 statistical analysis [5] identified 3409, 7506 and 8589 differentially expressed genes (DEGs) at 3, 6 and 10 days after sowing, respectively (Table S1).   Benjamini-Hochberg score < 0.05) between healthy and A. brassicicola infected conditions at 3, 6 and 10 days after sowing. Also showing shared DEGs among conditions at the pre-germinative stage (3 days) compared to the stages of seedling establishment (6 days) and autotrophy (10 days) of the seedling.
A Venn diagram comparison of the three developmental stages ( Fig. 3 ) exhibited 1192 common DEGs.

Plant Material
Arabidopsis (Col-0 ecotype) mature seed lots were obtained from plants grown in a controlled climatic room at 19/20 °C, 16 h photoperiod of artificial light (150 μmol photons m 2 s −1 ) and 70% relative humidity. Seeds (12 mg) were surface sterilised using 1 mL of 30% bleach treatment during 7 min, then followed by 7 min in 1 mL of 80% ethanol and five rinses in 1 mL of sterile deionized water. The seeds were dried for 5 h on a blotting paper in a Microbiological Safety Cabinet (SafeFAST Premium, FASTER, Cornaredo, MI, Italy).

Infection Assays
To select specific seed responses involved in the biotic interaction and not related to a germination defect, the seed inoculum concentrations were optimized to reach a maximal seed germination rate (Gmax). The Gmax as well as the infection rate of seeds of Arabidopsis ecotype Col-0 were evaluated to different concentrations of Abra43 A. brassicicola strain inoculum, i.e. 0, 10 2 , 10 3 , 10 4 , 10 5 conidia/mL, respectively.

Germination Assays
For seed inoculation, 1 mL of the solution at the appropriate conidia concentration was added for one hour to 15 mg of seeds. The inoculated seeds were dried for 5 h on a blotting paper in a Microbiological Safety Cabinet (SafeFAST Premium, FAST-ER). Seed germination analyses were performed in microplates using the ScreenSeed automate according to the conditions described by Merieux et al. [1] . Incubation was performed inside a thermo-regulated incubator (Memmert ICP 750) regulated at 22 °C ( ±1 °C). Four replicates were measured in each condition analyzed and a minimum of 100 seeds per repeat was analyzed.

Sample Preparation
All sterilized seeds were inoculated with 10 4 conidia/mL of A. brassicicola . The non-inoculated seeds were used as a control. Seeds infected or treated with water (non infected control seeds) were sowed in petri dishes containing 0.8% agarose (SIGMA) and cultures were incubated in a controlled growth chamber for 3, 6 and 10 days under a 16 h photoperiod (170 μmol photons m 2 s −1 ) at 22 °C (light period)/20 °C (dark period) and a constant 70% relative humidity. 20 mg of seeds were used for each sample with three biological replicates per condition.

RNA Extraction and Sequencing
Seeds were collected at 3, 6 and 10 days after sowing. RNA extraction was performed using NucleoSpin ® RNA Plus kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions. RNA quantification and quality were measured with a NanoDrop ND-100 (NanoDrop Technologies, DE, USA) and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) respectively. RNA samples were sent to Beijing Genomics Institute (BGI, https://www.bgi.com ), Hong Kong for cDNA library construction paired-end sequencing (PE100, 40M) and sequencing using a DNA nanoball sequencing (DNBSEQ TM ) technology. DNBSEQ TM technology performed by BGI sequencing platform includes the single strand circular library construction, DNB generation and loading method, cPAS (combinatorial Probe Anchor Synthesis) sequencing technology.

Ethics Statements
This work does not contain any studies with human or animal subjects .

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability
Dual-transcritome analysis of germinating Arabidopsis seeds in response to necrotrophic fungus Alternaria brassicicola (Original data) (NCBI GEO). de la Loire, Angers Loire Métropole and the European Regional Development Fund. We would like to thank to Adriana Tofiño, Aida Vasco, and Luz Marina Melgarejo for productive discussion about this project. Thanks to the FUNGISEM team for their support during the development of the investigation and to Lotta Grappin for vector drawings of the Fig. 2 .

Supplementary Materials
Supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.dib.2022.108530 .