A first insight into the involvement of phytohormones pathways in coffee resistance and susceptibility to Colletotrichum kahawae

Abstract

Understanding the molecular mechanisms underlying coffee-pathogen interactions are of key importance to aid disease resistance breeding efforts. In this work the expression of genes involved in salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) pathways were studied in hypocotyls of two coffee varieties challenged with the hemibiotrophic fungus Colletotrichum kahawae, the causal agent of Coffee Berry Disease. Based on a cytological analysis, key time-points of the infection process were selected and qPCR was used to evaluate the expression of phytohormones biosynthesis, reception and responsive-related genes. The resistance to C. kahawae was characterized by restricted fungal growth associated with early accumulation of phenolic compounds in the cell walls and cytoplasmic contents, and deployment of hypersensitive reaction. Similar responses were detected in the susceptible variety, but in a significantly lower percentage of infection sites and with no apparent effect on disease development. Gene expression analysis suggests a more relevant involvement of JA and ET phytohormones than SA in this pathosystem. An earlier and stronger activation of the JA pathway observed in the resistant variety, when compared with the susceptible one, seems to be responsible for the successful activation of defense responses and inhibition of fungal growth. For the ET pathway, the down or non-regulation of ET receptors in the resistant variety, together with a moderate expression of the responsive-related gene ERF1, indicates that this phytohormone may be related with other functions besides the resistance response. However, in the susceptible variety, the stronger activation of ERF1 gene at the beginning of the necrotrophic phase, suggests the involvement of ET in tissue senescence. As far as we know, this is the first attempt to unveil the role of phytohormones in coffee-C. kahawae interactions, thus contributing to deepen our understanding on the complex mechanisms of plant signaling and defense.

Fig 1. Time-course of Colletotrichum kahawae (isolate Que2) infection in hypocotyls of resistant (Catimor 88) and susceptible (Caturra) coffee varieties.

3hpi and 6hpi–beginning of conidia germination and appressoria differentiation, respectively; 12hpi– 42% of melanized appressoria; 24hpi–almost 100% of melanized appressoria; 48hpi—fungal penetration and biotrophic growth; 72hpi–switch to necrotrophy (susceptible variety) and restriction of fungal growth (resistant variety). Times after inoculation (bold) were selected to collect samples for gene expression studies by qPCR.

Fig 2. Fungal pre-and post-penetration growth stages and host responses.

Light microscope observations, cotton blue lactophenol staining (2a-2e) and epifluorescence test under blue light (2f). Fig 2a. Conidia (c) germination and formation of melanized appressoria (A) on the surface of a resistant hypocotyl, 24 hours post inoculation (hpi). Fig 2b. Infection site showing a melanized appressorium (A) and an infection vesicle (v) in the epidermal cell of the resistant hypocothyl, 48hpi. Fig 2c. Infection site showing a melanized appressorium (A) and hyphae inside two adjacent epidermal cells of the susceptible hypocotyl (arrow), 48hpi. Fig 2d. Fungal hyphae (arrows) in living and in necrotized (n) cells of the susceptible hypocotyl, 72hpi. Fig 2e. Infection site showing a melanized appressorium (A) and intracellular hyphae (arrow) confined to the epidermal cell of the resistant hypocotyl, 72hpi. Fig 2f. Infection site showing an appressorium (A) associated with autofluorescence of the cytoplasmic content of one epidermal cell (HR-like). Note that the walls of this cell and of adjacent epidermal and cortex cells are also autofluorescent (bars = 10μm).

Fig 3. Percentage of infection sites with host responses.

Accumulation of phenolic-like compounds in the cell walls only, or in both the cell walls and the cytoplasmic contents and deployment of hypersensitive-like cell death (HR) induced by C. kahawae in hypocotyls of coffee varieties Catimor 88 (R-resistant) and Caturra (S-susceptible), at different hours post inoculation. The average percentages were significantly higher in the resistant than in the susceptible coffee variety at 24hpi (t = 2.52; p ≤ 0.05), 48hpi (t = 4.83; p ≤ 0.001) and 72hpi (t = 6.69; p ≤ 0.001).

Fig 4. Expression analysis of SA, JA and ET pathway related genes in non-inoculated hypocotyls of Catimor 88 (R-resistant) and Caturra (S-susceptible) vs inoculated hypocotyls with C. kahawae.

Heatmap was colored according to the log2 ratio of expression, where green indicates lower expression, red indicates higher expression and black indicates no expression (see the color scale); in columns are the time-point studied (6, 12, 24, 48 and 72hpi) and in the rows the genes analyzed.

Fig 5. qPCR expression analysis of JA pathway related genes.

Relative expression patterns of OPR3 (biosynthesis), COI1 (receptor) and PR10 (responsive gene) obtained in Catimor 88 (R-resistant) and Caturra (S-susceptible) coffee varieties. Mean and standard deviation of three biological replicates is presented. Fold change as relative expression of gene expression between inoculated and control samples for each of the coffee varieties/inoculation time-points. Asterisks (∗) represent statistical significance (p≤0.05) of gene expression between the two coffee varieties was determined by the non-parametric Mann–Whitney U test using IBM^®SPSS^® Statistics version 20.0 (SPSS Inc., USA) software.

Fig 6. qPCR expression analysis of ET pathway related genes.

Relative expression patterns of ACS5/ACSO2 (biosynthesis), ETR1/EIN2 (receptors), CTR1 (negative regulator) and ERF1 (responsive gene) were obtained in Catimor 88 (R- resistant) and Caturra (S-susceptible) coffee varieties. Mean and standard deviation of three biological replicates is presented. Fold change as relative expression of gene expression between inoculated and control samples for each of the coffee varieties/inoculation time-points. Asterisks (∗) represent statistical significance (p≤0.05) of gene expression between the two coffee varieties was determined by the non-parametric Mann–Whitney U test using IBM^®SPSS^® Statistics version 20.0 (SPSS Inc., USA) software.