Comparative transcriptional analysis of Persea americana MYB, WRKY and AP2/ERF transcription factors following Phytophthora cinnamomi infection

Abstract

Plant cells undergo extensive transcriptional reprogramming following pathogen infection, with these reprogramming patterns becoming more complex when pathogens, such as hemibiotrophs, exhibit different lifestyles. These transcriptional changes are often orchestrated by MYB, WRKY and AP2/ERF transcription factors (TFs), which modulate both growth and defence-related gene expression. Transcriptional analysis of defence-related genes in avocado (Persea americana) infected with Phytophthora cinnamomi indicated differential immune response activation when comparing a partially resistant and susceptible rootstock. This study identified 226 MYB, 82 WRKY, and 174 AP2/ERF TF-encoding genes in avocado, using a genome-wide approach. Phylogenetic analysis revealed substantial sequence conservation within TF groups underscoring their functional significance. RNA-sequencing analysis in a partially resistant and susceptible avocado rootstock infected with P. cinnamomi was indicative of an immune response switch occurring in either rootstock after 24 and 6 h post-inoculation, respectively. Different clusters of co-expressed TF genes were observed at these times, suggesting the activation of necrotroph-related immune responses at varying intervals between the two rootstocks. This study aids our understanding of avocado immune response activation following P. cinnamomi infection, and the role of the TFs therein, elucidating the transcriptional reprogramming disparities between partially resistant and susceptible rootstocks.

Keywords: Phytophthora; ERF; MYB; WRKY; avocado; defence‐related genes; transcription factor.

FIGURE 1

Gene and protein structures of 226 MYB sequences from the Persea americana 'West‐Indian' pure accession genome. (a) Phylogenetic analysis was done using the neighbour‐joining method, Jones–Thornton–Taylor (JTT) method and 1000 bootstrap following ClustalW alignment of PaMYB sequences. (b) Conserved motifs identified within MYB protein sequences using the MEME Suite online tool, with sequences corresponding to the indicated blocks being listed on the right of the figure. The conserved sequences were used to divide the sequences into different MYB subfamilies. (c) Exon–intron boundaries of the MYB genes, with gene sizes indicated in kilobase pairs (kb). Exons are represented by yellow boxes, introns by black lines and untranslated regions (UTRs) by green boxes (R2R3—R2R3‐MYB; R1, R2 and R3—1R‐MYB).

FIGURE 2

Conserved WRKY motifs and gene exon–intron boundaries from the Persea americana 'West‐Indian' pure accession genome. (a) A neighbour‐joining tree constructed using the Jones–Thornton–Taylor (JTT) method, with 1000 bootstrap replications, following ClustalW alignment of PaWRKY sequences. (b) The MEME online tool was used to identify 15 conserved motifs (sequences shown on the right of the figure) within 82 WRKY protein sequences. These motifs were used to assign WRKY proteins into different groups and subgroups, which are shown on the right in the phylogenetic tree. (c) WRKY gene exon–intron regions, with gene sizes indicated in kilobase pairs (kb). Yellow boxes indicate exons, green boxes indicate untranslated regions and black lines indicate introns.

FIGURE 3

Conserved motifs of AP2/ERF family proteins from the Persea americana 'West‐Indian' pure accession genome, together with gene exon–intron boundaries. (a) A neighbour‐joining phylogenetic tree constructed using the Jones–Thornton–Taylor (JTT) method, with 1000 bootstrap replications, following ClustalW alignment of PaAP2/ERF sequences. (b) Fifteen motifs within 174 sequences were identified using the MEME online tool, with the motif sequences being listed on the right of the figure. (c) Exon–intron boundaries of AP2/ERF‐encoding genes, with gene sizes indicated with kilobase pairs (kb). Green boxes represent exons, yellow boxes represent untranslated regions and black lines represent intron regions.

FIGURE 4

MYB transcription factor expression patterns in partially resistant Dusa and susceptible R0.12 avocado rootstocks following Phytophthora cinnamomi inoculation. (a) Heatmap showing the log₂fold‐change (log₂FC) in the expression of 118 MYBs in infected Dusa and R0.12 rootstocks, with mock‐inoculated samples set as the reference. Cells with cross symbols indicate a significant difference in expression levels (log₂FC ≥ |1|, p ≤ 0.05). A positive log₂FC (red) indicates higher expression levels compared to that in mock‐inoculated samples, and a negative log₂FC (blue) indicates lower expression levels. (b) Heatmap showing the difference in the expression level (as log₂FC) of 73 MYBs when Dusa samples are compared with R0.12 samples set as reference following P. cinnamomi inoculation. A positive log₂FC (red) value indicates the level of expression is higher in Dusa compared to R0.12, and a negative log₂FC (blue) value indicates higher expression levels in R0.12. Cross symbols indicate significant log₂FC values (MI, mock‐inoculated).

FIGURE 5

Expression level differences of WRKY genes in two avocado rootstocks following Phytophthora cinnamomi inoculation. (a) Heatmap of the differences in expression levels as log₂fold‐change (log₂FC) in Dusa (partially resistant) and R0.12 (susceptible) rootstocks at different time points (hours post‐inoculation; hpi) following inoculation. Mock‐inoculated samples are set as the reference, thus positive log₂FC values (red) indicate higher expression levels in inoculated samples, and negative log₂FC values (blue) indicate lower expression levels in inoculated samples. (b) Heatmap showing the level of WRKY expression compared between the two rootstocks, with R0.12 set as the reference. Positive log₂FC values (red) indicate higher WRKY expression levels in Dusa and negative log₂FC values (blue) indicate higher levels of expression in R0.12. (+ symbols indicate significance, log₂FC ≥ |1|, p ≤ 0.05; MI, mock‐inoculated).

FIGURE 6

The level of expression of AP2/ERFs transcription factors in partially resistant Dusa and susceptible R0.12 avocado rootstocks following Phytophthora cinnamomi inoculation. (a) Heatmap showing expression differences of 116 AP2/ERFs (as log₂fold‐change values; log₂FC) compared to mock‐inoculated samples. (b) Heatmap of 87 AP2/ERFs genes showing significant differences in expression level following P. cinnamomi inoculation when compared between the two rootstocks, with R0.12 set as the reference. Log₂FC values larger than zero (red) indicate higher expression levels in Dusa, and log₂FC values smaller than zero (blue) indicate higher expression in R0.12. (+ symbols indicate significance, log₂FC ≥ |1|, p ≤ 0.05; MI, mock‐inoculated).