The inbred maize lines SUS (IL09) and RES (IL02) were used, whose contrasting responses to Fv root rot were previously evaluated (Román, 2017; Roman et al., 2020). The highly virulent Fv strain DA42 was previously characterized by Leyva-Madrigal et al. (2015) and was used to conduct the infection experiments.

Previously described protocols were used for seedling preparation (Warham et al., 1996; Román, 2017; Roman et al., 2020). Briefly, the seeds were superficially disinfected by sonication (2.8 L Ultrasonic Bath, Fisher Scientific) in sterile distilled water with Tween 20 (five drops of Tween 20/100 ml of distilled water) for 5 min. Subsequently, the seeds were immersed in 1.5% (V/V) sodium hypochlorite at 52°C for 20 min in a thermobath (FE-377, Felisa) and rinsed three times in sterile distilled water under sterile conditions in a biological safety cabinet (Herasafe KS, Thermo Scientific). The fungus was cultured in Spezieller Nährstoffarmer agar medium (SNA) (Leslie and Summerell, 2007) with 1 cm² filter paper at 25 ± 2°C for 14 days. Conidia were harvested by adding 5 ml of sterile saline solution (0.8% NaCl) to the culture medium with gentle shaking. The conidia working aqueous solution was prepared at a final concentration of 1 × 10⁶ conidia/ml by quantification in a Neubauer chamber (cat. No. 3110, Hausser Scientific, USA) using a light microscope (B-383-M11, Optika, Italy). Disinfected seeds were immersed for 5 min in the conidia work solution, whereas control seeds were immersed in sterile water. Subsequently, 10 seeds were distributed, each 2 cm thick, on sterile Kraft paper (40 cm length × 20 cm width), moistened with sterile water, rolled up, and placed in plastic bags. The seeds germinated in a 16:8 h light:dark photoperiod for seven to 10 days at 25°C. The humidity of the rolls was maintained by irrigating them with 15 ml of water every 24 h. Visual records and photographs were taken every day with a stereo microscope (model M205FA, Leica, Germany).

Root RNA was extracted at seven days old after seed germination for both RES (uninfected and infected) and SUS (uninfected and infected) genotypes. Total RNA extraction was performed using the RNeasy^® Plant Mini Kit (Qiagen, Cat. No. 74904), according to the manufacturer. For library preparation and sequencing, 500 ng of RNA were used as input material for each RNA sample preparation. Twelve libraries (three independent biological replicates) per analyzed condition (uninfected and infected plants) from the SUS and RES genotypes were generated. The TruSeq RNA Sample Preparation Kit (Illumina, Cat. N°RS-122-2002) was used following the manufacturer’s recommendations, and index codes were utilized to identify each sample independently. The libraries were sequenced on the NextSeq 500 platform (Illumina) using the 2 × 150 bp paired-end sequencing protocol in the facilities of LANGEBIO (langebio.cinvestav.mx). The bioinformatic analysis was conducted on the OOREAM server of IPN, CIIDIR Sinaloa. The quality of reads was examined with FastQC v0.11.7 (www.bioinformatics.babraham.ac.uk/projects/fastqc/) before and after the trimming process. Raw reads were filtered with Trimmomatic 0.36 (Bolger et al., 2014) to eliminate adapters, low-quality reads (Q <20), and short reads (<50 bp). Trimmed reads were pseudo-aligned with Kallisto v0.46.2 (Bray et al., 2016) to the reference transcriptome of Z. mays cv. B73 v5 (www.maizegdb.org). The transcript abundance files were imported into R v4.2.2 with the package tximport v1.26.1, and then differential expression analysis was conducted with the package DESeq2 v1.38.3 (Love et al., 2014). Differentially expressed genes (DEGs) were defined as those genes with an adjusted P-value <0.05 and a log2 fold change ±1. DEGs were subjected to KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) enrichment analyses with the packages clusterProfiler v4.6.2 (Wu et al., 2021) and REVIGO (Supek et al., 2011), respectively.

Primary root tissue close to the seed (the first 0.5 cm) was harvested for light microscopy and scanning electron microscopy (SEM) studies. For light microscopy, tissues were fixed in 96% ethyl alcohol and stored at 4° C. Tissues were then placed in a histocassette and gradually dehydrated in ethyl alcohol. Subsequently, they were placed in a 1:1 solution of ethyl alcohol and 100% xylol (Spintissue Processor STP120 and Histo Star Embedding Centre, Thermo Scientific). The dehydrated samples were embedded in Histoplast (cat. no. 22900700, Fisherbrand), and 5 μm-thick cross-sections were cut with a microtome (Microm HM 340E, Thermo Scientific) and incubated in a drying oven (DX402, Yamato) at 60° C for 30 min, then placed in xylene for 2 min to remove paraffin. Tissue samples were then rehydrated in 1× PBS and visualized for endogenous fluorescence by epifluorescence microscopy (DIM6000, Leica, Germany). For SEM, the samples were treated as previously described by Olivares-Garcia et al. (2020).

RNA was extracted from RES (C and +Fv) and SUS (C and +Fv). Each sample was integrated with the root tissue from 20 plants, and the tissue was macerated in liquid nitrogen. Total RNA isolation was performed using TRIzol™ (15596018, Ambion, CA) according to the manufacturer’s specifications. The cDNA was synthesized using 1,000 ng of RNA treated previously with DNAse (AM2222, Invitrogen) using the Super Script™ IV First Strand Synthesis System kit (18091050, Invitrogen) according to the manufacturer’s protocol. A ddPCR (droplet digital PCR) was performed according to the manufacturer’s instructions; briefly, the Supermix for EvaGreen Qx200TM was used, and the reaction was carried out in a final volume of 20 µl with final concentrations of 1× Qx200, 100 nM for each primer, and 50 ng of cDNA. The drops were generated in droplet generator equipment, and these were recovered and transferred to a 96-well ddPCR plate (BIO-RAD, USA). The PCR protocol consisted of one cycle of enzyme activation at 95°C for 5 min; 40 cycles of denaturation at 95°C for 30 s; alignment and extension at 60°C for 1 min with an increase of 2°C every second; and one signal stabilization cycle consisting of 4°C for 5 min and then 90°C for 5 min. And in the end, a storage temperature of 4°C. The reaction was carried out in a T100™ thermal cycler (BIO-RAD, USA). At the end of the protocol, the drops were read on the QX200™ Droplet Reader (BIO-RAD, USA). Three technical replicas for each independent biological replicate were used for the quantification assay. The result was reported in copies per µl (copies/µl), and the statistical analysis and the graphics were performed by OriginPro 8.5 software (developed by OriginLab Corporation, Northampton, USA), using the statistical Tukey test with a significance of 0.05. Primers were designed for peroxidases (XM_020546021, Fw-CCGAGGACATCATCAAGCAA and Rv-GAGTTGATGAGGATGGAGCC), cellulose synthases (XM_035960774.1, Fw-CGCTGGATTTGACGACGA and Rev-AGGAACACCACCATACTCCA), and expansin (EU960208.1, Fw-TTTTCTCCTCCCCATCCAGT and Rev-CTTCACGGAGGCACTTAACA) involved in cell wall. The design was carried out using the Primer3plus software with parameters suggested for the dd PCR.

Raw data from the microscopy studies were analyzed to determine if they differed from a normal distribution (Shapiro–Wilk test). Variables that conformed to parametric assumptions were analyzed using one-way analysis of variance (ANOVA) and Duncan’s means test (α <0.01); those that did not were analyzed using the non-parametric Kruskal–Wallis test and Dunn’s pairwise comparison test. The experiment was repeated three times independently, with three replicates (n = 15) for microscopy analyses. All statistical analyses were conducted in R v4.2.2. Origin v8.5.1 and CorelDraw v20.0.0.633 were used to generate graphs and figures, respectively.

An infection time course was performed to investigate the Fv infection (+Fv) process in RES and SUS genotypes and to determine the optimal day to visualize sample root tissue for transcriptomics analysis ( Figure 1 ). The color of the pericarp turned purple in the infected seeds of RES and SUS, and the Fv mycelium was visualized by stereomicroscopy three days after seed inoculation (DAI). However, the mycelium was visible until four DAI ( Figure 1A , arrowheads). Mycelium abundance was more evident in SUS than in RES, and the pericarp displayed necrotic spots only in SUS ( Figure 1A , circle). At four to five DAI, small necrotic spots were visible on SUS seed roots, and seed rot commenced, whereas necrotic spots were observed on RES between six and seven DAI. Necrotic spots on secondary roots began at five DAI in SUS. In contrast, RES only showed a color change up to eight DAI in the same tissue. SUS seed rot began at five or six DAI, becoming evident at eight DAI ( Figure 1B ). Interestingly, seed rot in RES was observed at 12 DAI (data not shown), demonstrating that seven DAI is the most suitable time for the analyses.

Gene expression during Fv root infection in RES and SUS genotypes was analyzed by RNA-Seq. Since Fv induces necrosis in infected tissues, seven DAI were selected for RNA isolation from root tissue to preserve the highest possible RNA integrity and quality. Only samples with RNA integrity numbers between 7.9 and 9.9 were considered for library construction and sequencing. Three biological replicates for each condition were obtained, but one atypical replicate per condition was removed according to principal component analysis ( Supplementary Figure S1A ). On average, 83.2 and 76.2 million trimmed reads per library were kept for RES (C and +FV) and SUS (C and +FV) genotypes, respectively ( Supplementary Table S1 ). The differential expression analysis in response to +Fv infection ( Supplementary Tables S2 , S3 for RES and SUS, respectively) showed 798 and 624 DEGs for RES and SUS genotypes, respectively. From those, 733 and 559 DEGs were exclusive for RES and SUS, respectively, and only 65 DEGs were common in both genotypes. In RES, 294 DEGs (40%) were upregulated and 439 DEGs (60%) were downregulated. While in SUS, 258 DEG (46%) were upregulated and 301 DEGs (54%) were downregulated ( Supplementary Figure S2 ). The expression profiles of DEGs for RES and SUS genotypes were visualized in heatmaps ( Supplementary Figures S1B, C ), showing similar profiles between the two replicates of each condition.

To identify pathways involved in Fusarium infection, we performed a KEGG enrichment analysis. At a p-value threshold of ≤0.05, several pathways were highlighted in the two comparisons ( Figure 2 and Table 1 ). In general, RES genotype showed metabolic pathway enrichment to produce specific metabolites, i.e., coumarin and furanocoumarin, phenylpropanoids, and transcripts related to plant-pathogen interactions (GeneRatio = 1). In contrast, only SUS showed positive enrichment for transcripts involved in membrane rearrangements (endocytosis), and a negative enrichment for pathways involved in photosystems and the plant–pathogen interaction pathway, among others (GeneRatio >0.8). Specific transcripts involved in the KEGG analysis ( Supplementary Table S4 ) indicate that several transcripts are related to cell wall proteins (pectinesterase, phenylalanine ammonia-lyase, beta-amylase, and peroxidase). In addition, the RES genotype exhibits enrichment in the phenylpropanoid biosynthesis pathway, which contains transcripts related to cell wall biogenesis.

A more detailed analysis of the DEG was conducted to identify the GO terms in abundance ( Figures 3A, B ). This analysis made it possible to identify clustered transcripts related to proteins in the same pathways, which were also previously detected in KEGG enrichment analysis. In general, GO term abundance analysis revealed transcripts related to secondary metabolism, phenylpropanoids, lignin, cell wall processes, plant–pathogen interactions, and hormone signal transduction in maize RES and SUS genotypes. Interestingly, some cell wall transcripts related to cell wall organization biogenesis, cell wall organization, cell wall biogenesis, pectin biosynthesis, and cellulose biosynthesis were also detected. Attention was therefore focused on transcripts related to the cell wall that were identified in the transcriptomes of both maize genotypes.

Twenty-one transcript IDs in the RES genotype and eleven transcript IDs in the SUS genotype were identified as cell wall-related transcripts ( Figures 3C, D ). Some of these transcripts were also identified in the previous KEGG enrichment analysis, specifically peroxidase (A0A1D6K433), UDP-glycosyltransferase (A0A1D6HWI3), beta-amylase (B6SYP0), and pectinesterases (A0A3L6FHM0 and B8A2X5). The RES genotype shows a higher number of expressed cell wall-related transcripts as compared to the SUS genotype. RES genotype also has a high percentage (47%) of cell wall transcripts that are highly expressed in relation to the control condition, whereas SUS genotype shows a high percentage (64%) of downregulated cell wall-related transcripts relative to the control condition ( Figures 3C, D ). DEG transcripts putatively related to lignin metabolism, including peroxidases and laccases, were upregulated (1.7 to 7.2 log2 fold change) in RES genotype, whereas the phenylalanine ammonia-lyase transcript was surprisingly downregulated (−23.1 log2 fold change) in the SUS genotype. Interestingly, another cell wall remodeling enzyme, beta-amylase, was upregulated (9.9 log2 fold change) in the RES genotype, whereas it was downregulated (−10.5 log2 fold change) in the SUS genotype. In addition, pectinesterases were strongly downregulated (−23.8 Log2 FC) in the SUS genotype. Finally, beta-amylase and several pectinesterases were strongly reduced in the SUS genotype, suggesting cell wall disorganization in this genotype, whereas the RES genotype displayed more transcriptional activity than the SUS genotype among genes related to cell wall dynamics.

To examine the potential role of the cell wall in maize roots during Fv infection, histological sections were made from the first 5 mm of the primary root at the pedicel zone and visualized by endogenous fluorescence ( Figure 4 ). The spatial distribution of fluorescence was slightly higher in the SUS genotype than in the RES genotype ( Figure 4B ). However, the RES genotype fluorescence was increased in the first cellular layers of the epidermis–hypodermis zone during Fv infection ( Figure 4A , arrowheads). In contrast, the SUS genotype presented a more homogenous fluorescence along with more fragile cellular tissues during histological manipulation ( Figure 4A , asterisks), which could compromise the epidermis–hypodermis integrity during Fv attack. The average endogenous fluorescence decreased in both RES and SUS genotype tissues during Fv infection in comparison to the control, although no statistical differences were observed. The SUS genotype displayed a higher fluorescence in both control and infected samples than the RES genotype ( Figure 4B ). To study the first cellular layer (epidermis), which is the first site to be targeted by Fv attacks, the primary roots of uninfected and infected tissues were observed by SEM ( Figure 5 ). In the uninfected conditions, RES root hairs were turgid and there was a homogenous distribution of epidermis cells, whereas the root hairs in SUS were not turgid, and SEM of SUS epidermis cells revealed small holes between cell–cell junctions, probably due to an abnormal distribution in the cells ( Figure 5 , arrowheads). In infected roots, SUS root hairs and the epidermis cell wall were degraded by Fv infection, whereas in RES, the Fv mycelium could be visualized between the root hairs ( Figure 5 , arrows), and cells were slightly turgid but not collapsed. Taken together, these results suggest that the cell walls of the first cellular layers in maize roots play an important role during Fv infection. Lignin content and confocal microscopy analysis (manuscript in preparation) have revealed a spatial distribution of lignin (stained with basic fuchsin) similar to that observed by endogenous fluorescence, which was correlated with the increased signal in sclerenchyma cells (hypodermis). These findings suggest that the cell wall of the first cell layers at the epidermis–hypodermis zone may have an active role against Fv infection due to the strengthening of the cell wall by lignification.

The gene expression levels of the three-cell wall-related genes evaluated were higher in RES than in SUS during Fv infection ( Figure 6 ). In the control, Prx gene expression was higher in SUS than RES; however, the expression has increased in RES, contrary to SUS, where its expression was downregulated in roots infected by Fv. For Cesa, in both RES and SUS genotypes, expression was closed, whereas Cesa expression was stimulated by Fv infection; suppressively, SUS root cells did not change their expression levels. This same behavior in gene expression was observed when expansin expression was evaluated. These observations suggest that the root cell wall in RES has a better dynamic to protect against Fv infection.