Genome-Wide Transcriptional Response of Avocado to Fusarium sp. Infection

Abstract

The avocado crop is relevant for its economic importance and because of its unique evolutionary history. However, there is a lack of information regarding the molecular processes during the defense response against fungal pathogens. Therefore, using a genome-wide approach in this work, we investigated the transcriptional response of the Mexican horticultural race of avocado (Persea americana var. drymifolia), including miRNAs profile and their possible targets. For that, we established an avocado-Fusarium hydroponic pathosystem and studied the response for 21 days. To guarantee robustness in the analysis, first, we improved the avocado genome assembly available for this variety, resulting in 822.49 Mbp in length with 36,200 gene models. Then, using an RNA-seq approach, we identified 13,778 genes differentially expressed in response to the Fusarium infection. According to their expression profile across time, these genes can be clustered into six groups, each associated with specific biological processes. Regarding non-coding RNAs, 8 of the 57 mature miRNAs identified in the avocado genome are responsive to infection caused by Fusarium, and the analysis revealed a total of 569 target genes whose transcript could be post-transcriptionally regulated. This study represents the first research in avocados to comprehensively explore the role of miRNAs in orchestrating defense responses against Fusarium spp. Also, this work provides valuable data about the genes involved in the intricate response of the avocado during fungal infection.

Keywords: Fusarium infection; Persea americana; defense response; miRNAs.

Figure 1

Symptoms of fusariosis in seedlings of avocado var. drymifolia at 30 dpi. Photography triptych on the left: leaf, stem, and root of uninfected plants (Control). Photography triptych on the right: leaf, stem, and root of infected seedlings. Infected plants were root inoculated with 1 × 10⁶ water-conidia suspension; control plants were treated with sterile water.

Figure 2

Schematic representation of some relevant genome metrics of avocado var. drymifolia genome. (a) Available assembled genome harbor 822.49 Mbp contained in a total of 7159 scaffolds. Input data from generating this new version were previously reported from Rendón-Anaya et al. [34] and were downloaded from GenBank. In total, 60.77% of the whole genome sequence (totaling 822.49 Mbp) was successfully anchored to the genetic map. A pie chart was used to visualize this information. (b) The gene set, which was predicted in both anchored and not anchored genomic sequences, comprises a total of 36,200 genes (25,959 and 10,241, respectively). (c) The anchored genome sequences to the genetic map are shown in a chromosome-scale graph. (d) Completeness estimated based on single copy orthologs shared between flowering plants from the dicotyledon clade (n = 1375). The bar’s colors represent the classes resulting from the BUSCO assessment.

Figure 3

Genes of avocado var. drymifolia identified as differentially expressed (DE) in response to Fusarium sp. infection. (a) Heatmap of expression profiles showing differentially expressed genes (DEGs), (b) Hierarchical clustering tree that shows closeness (or similarity) between the distinct sampling points included in differential expression analysis, (c) Venn diagram which show DEGs identified on each sampling point. In parentheses, the percentage of the total represented by those DEGs shared or not, between each sampling point.

Figure 4

Clusters of DEGs formed based on their expression profiles and GO enrichment analysis, which shows the most representative functional categories for each cluster (six in total; C1–C6, respectively). (a) Clusters of DEGs with similar expression patterns responsive to Fusarium sp. infection. (b) Representative biological processes for each cluster generated).

Figure 5

Main enriched hormonal processes in response to Fusarium sp. The figure shows the main phytohormones involved in the pathogenesis process and the number of genes involved in the regulated associated processes, which are biosynthesis, metabolism, transport, signaling, and regulation of SAR responses. Gray bars show those genes shared in multiple biological processes.

Figure 6

DEmiRNAs responsive to Fusarium sp. infection and the biological processes (BP) regulated by them. (a) UpSet plot of identified DEmiRNAs representing the number of target genes associated with each of them. (b) Bubble plot representing the main BP in which the associated targets of each identified DEmiRNA could intervene. The figure in the (b) panel was generated only considering the annotated target genes.

Figure 7

Involvement of DEmiRNAs in phytohormone regulation. The bubble plot illustrates the primary phytohormones and their respective roles, including biosynthesis, transport, metabolism, and involvement in SAR responses. It also indicates how the identified DEmiRNAs might intervene in these processes.

Figure 8

A schematic representation of the innate immune system model by which avocado var. drymifolia seeks to counteract the Fusarium sp. infection. In avocado defense responses initiated after Fusarium sp. recognition. Genes associated with signal transduction activation, which are responsive to the recognition of elicitor molecules, such as LYK3 and RLK1, were evidenced. The recognition is also mediated by R genes, exemplified by RPP13. This recognition and signaling cascades allow the accumulation of reactive oxygen species (ROS) and the activation of various transcription factors (TFs). Activation of these TFs facilitates the involvement of four main processes: microRNA expression, phenylpropanoids biosynthesis, biosynthesis and involvement of phytohormones, and expression of different genes. The main phytohormones involved in pathogenesis responses are AUX, ET, JA, SA, and ABA, the latter being the most represented in hormone-mediated signaling process. ABA can negatively regulate ET and AUX activity by suppressing genes such as YUCCA4 and EIN3 and intervene in JA signaling by regulating the MYC2 gene. ET and JA act synergistically with the involvement of WRKY33 gene. AUX, on the other hand, is involved, like phenylpropanoids biosynthesis, in root development, where AUX transporter activity is represented by ABC, PIN, and AUX. One process represented is SAR response, which is mediated by crosstalk between phytohormones such as ET, JA, and SA, in addition to the involvement of different genes considered important for optimal SAR responses, such as ELP2, FLD, FVE, and FMO1, as well as transporters like EDS5, highlighting the importance of this process as a primary response during the pathogenesis event. The regulatory involvement of microRNAs is reflected at different levels of regulation process. They can intervene in pathogen recognition regulation, as in the case of the miRNA/gene pair miR157/LYK3 and Ctg0854_RaGOO_5920/RLK1. They can also be involved in phenylpropanoids biosynthesis, as in the case of miR166/MYB4, and regulate AUX activity with the action of chr11_RaGOO_17754 on the YUC5 gene. Both miR166b and chr11_RaGOO_17754 may regulate root development. Finally, SAR may be regulated by the activity of miR166 on the JAR1 gene and chr4_RaGOO_33952 on the PEN3 gene.