Fig 1.
Post-infestation development of physiological and aerial symptoms.
Variation in net assimilation rates (AN; A and B), stomatal conductance (gs; C and D), transpiration rate (E; E and F) and intrinsic water use efficiency (AN/gs; G and H) in ‘Dusa’ and BG83 avocado plants at stage 1 (no aerial symptoms) after inoculation with R. necatrix. The asterisks indicate significant differences between inoculated and control treatments (P<0.05). Each data point represents the mean (±SE; n = 8 to 18). The arrows indicate root sampling for RNA extractions.
Fig 2.
Venn diagram of differentially expressed genes.
Numbers of common and specific differentially expressed genes (DEGs) obtained in the microarray analysis of susceptible ‘Dusa’-b, ‘Dusa’-a and tolerant BG83 avocado rootstocks after infection with R. necatrix. Shared transcripts are illustrated where the circles meet, while unique DEGs are shown in only one of the three circles. A higher number of both specific and shared DEGs, were observed in the susceptible ‘Dusa’ than in the tolerant BG83 rootstock.
Table 1.
qRT-PCR and microarray expression data of selected contigs from susceptible ‘Dusa’-b, ‘Dusa’-a and tolerant BG83 avocado rootstocks infected with R. necatrix.
Fig 3.
Principal component analysis (PCA) of differentially expressed transcripts (DEGs).
PCA of DEGs obtained in the microarray analysis of susceptible ‘Dusa’-b, ‘Dusa’-a and tolerant BG83 avocado rootstocks after infection with R. necatrix. PCA of DEGs present in at least one of the samples, in which the first two dimensions explain up to 88.74% of the variance. Samples from the tolerant BG83 avocado rootstock showed the largest differences with those from the susceptible ‘Dusa’(‘Dusa’-b, ‘Dusa’-a), which were grouped together in the biplot. Numbers are as follows: 1: protein hothead-like (Pa_Contig00205); 2: chitinase 1-like (Pa_Contig00535); 3: beta-glucanase (Pa_Contig00542); 4: non-annotated (NA) (Pa_Contig00559); 5: basic endochitinase-like partial (Pa_Contig01014); 6: thaumatin-like protein (Pa_Contig01450); 7: basic 7s globulin-like (Pa_Contig02817); 8: glucan endo-beta-glucosidase-like (Pa_Contig03461); 9: trypsin inhibitor (Pa_Contig04097); 10: protein hothead-like (Pa_Contig04808); 11: glu protease inhibitor-like (Pa_Contig05213); 12: protein hothead-like (Pa_Contig06176); 13: NA (Pa_Contig06358); 14: basic 7s globulin-like (Pa_Contig06808); 15: pathogenesis-related protein pr-4-like (Pa_Contig07140); 16: protein hothead-like (Pa_Contig07184); 17: NA (Pa_Contig07403).
Fig 4.
Hierarchical clustering (HCL) of differentially expressed genes (DEGs).
HCL of DEGs in at least one of the samples of ‘Dusa’-b, ‘Dusa’-a and tolerant BG83 avocado rootstocks after infection with R. necatrix, clustered by hclust according to their expression profiles (Pearson’s values correlation analysis). The five colours on the left correspond to the five groups with different expression profiles.
Table 2.
Top 10 avocado transcripts differentially regulated by R. necatrix in ‘Dusa’-b, ‘Dusa’-a and BG83.
Fig 5.
Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs).
GO enrichment analysis of DEGs in susceptible ‘Dusa’-b, ‘Dusa’-a and tolerant BG83 avocado rootstocks after infection with R. necatrix. Enrichment GO terms were obtained by Blast2GO using a cut-off of 0.025. (BP) biological process; (MF) molecular function; (CC) cellular component.
Table 3.
Defence-related genes overexpressed in ‘Dusa’-b, ‘Dusa’-a and BG83 avocado rootstock following R. necatrix infection.
Fig 6.
Comparative model of Rosellinia necatrix infection on susceptible ‘Dusa’ (A) and Tolerant BG83 genotypes (B). 1A. Inoculation of asymptomatic ‘Dusa´plant with wheat grains infected with R. necatrix. 2A. 8 days post inoculation. Visualization of R. necatrix derivative strain CH53-gfp mycelia colonizing the root surface of avocado plantlets without aerial symptoms. The green fluorescent emitted from R. necatrix-gfp was visualized using a fluorescence microscope. Upregulation of genes mainly related to oxidoreduction activity (Redox), cell wall degradation (CWD) as well as potential genes involved in defence to P. cinnamomi (PR) was observed. 3A. 22 days post inoculation. The image shows a radial section of susceptible roots infected with R. necatrix-gfp; penetration and proliferation of R. necatrix mycelia through the root cortex was observed by Confocal Laser Scanning Microscopy (CLSM) concomitant with a decrease in leaf photochemistry (Fv´/Fm´) and gas exchange parameters (AN, gs, E). Expression of genes related with cell wall degradation enzymes (CWD) mainly, chitin related enzymes, become more relevant. 4A. 30 days post inoculation, mild-wilting symptoms of the leaves start to appear in susceptible genotype. CLSM images of radial sections of the roots revealed a profuse invasion of R. necatrix, collapsing vascular vessels. 1B. Inoculation of asymptomatic BG83 plant with wheat grains infected with R. necatrix. 2B. 8 days post inoculation. Visualization of R. necatrix derivative strain CH53-gfp mycelia colonizing the root surface of avocado plantlets without aerial symptoms. 3B. 30 days post inoculation. Asymptomatic tolerant BG83 genotype showing upregulation of genes related to osmotic (OE) and salt stress (SE) as well as genes encoding protease inhibitors (PI).