Time-resolved dual transcriptomics reveal early induced Nicotiana benthamiana root genes and conserved infection-promoting Phytophthora palmivora effectors

Abstract

Background: Plant-pathogenic oomycetes are responsible for economically important losses in crops worldwide. Phytophthora palmivora, a tropical relative of the potato late blight pathogen, causes rotting diseases in many tropical crops including papaya, cocoa, oil palm, black pepper, rubber, coconut, durian, mango, cassava and citrus. Transcriptomics have helped to identify repertoires of host-translocated microbial effector proteins which counteract defenses and reprogram the host in support of infection. As such, these studies have helped in understanding how pathogens cause diseases. Despite the importance of P. palmivora diseases, genetic resources to allow for disease resistance breeding and identification of microbial effectors are scarce.

Results: We employed the model plant Nicotiana benthamiana to study the P. palmivora root infections at the cellular and molecular levels. Time-resolved dual transcriptomics revealed different pathogen and host transcriptome dynamics. De novo assembly of P. palmivora transcriptome and semi-automated prediction and annotation of the secretome enabled robust identification of conserved infection-promoting effectors. We show that one of them, REX3, suppresses plant secretion processes. In a survey for early transcriptionally activated plant genes we identified a N. benthamiana gene specifically induced at infected root tips that encodes a peptide with danger-associated molecular features.

Conclusions: These results constitute a major advance in our understanding of P. palmivora diseases and establish extensive resources for P. palmivora pathogenomics, effector-aided resistance breeding and the generation of induced resistance to Phytophthora root infections. Furthermore, our approach to find infection-relevant secreted genes is transferable to other pathogen-host interactions and not restricted to plants.

Keywords: De novo transcriptome assembly; Dual transcriptomics; Effectors; N. benthamiana; Non-model species; P. palmivora; RXLR effectors; Secretome.

Fig. 1

Phytophthora palmivora exerts a hemibiotrophic lifestyle in Nicotiana benthamiana roots. a Representative pictures of root-infected plantlets during P. palmivora infection, showing disease progression on the aboveground tissues. The successive symptom extent stages (SESs) were used to define a disease index in order to quantitate disease progression over time. b–h Microscopic analysis of N. benthamiana roots inoculated with transgenic P. palmivora LILI expressing an endoplasmic reticulum (ER)-targeted yellow fluorescent protein (YFP). Pictures were taken during penetration (b, 3 h after inoculation (hai)), early infection (c, 6 hai), biotrophy (d, 18 hai and e, 24 hai), switch to necrotrophy (f, 30 hai) and necrotrophy (g, 48 hai and h, 72 hai). Each panel shows transmission light (Transmission) and merged YFP fluorescence with propidium iodide (PI) staining (YFP + PI). Hy hypha, Ve vesicle, Cy cyst, Ha haustorium. Scale bar is 10 μm. i Quantification of P. palmivora biomass accumulation over time in N. benthamiana roots was measured by expression of P. palmivora WS21 relative to N. benthamiana L23 and F-box reference genes. j, k Expression of P. palmivora lifestyle marker genes Hmp1 (j) and Cdc14 (k) were measured over time relative to P. palmivora WS21 and OPEL reference genes. Quantitative RT-PCR experiments were performed in triplicate. Circles represent values for each replicate. Bars represent the mean value. Statistical significance has been assessed using one-way analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) test (p < 0.05)

Fig. 2

Overview of P. palmivora sequencing data analysis workflows. a Selection of P. palmivora reads from mixed samples and de novo assembly of transcriptome. b Secretome prediction. c Pipeline for automated secretome annotation. Final products of each pipeline are highlighted by bold lines. SP signal peptide, NLS nuclear localisation signal, CRN Crinkler

Fig. 3

N. benthamiana and P. palmivora transcriptomes show different temporal dynamics during interaction. a, b PCA clustering of full transcriptional profiles of N. benthamiana (a) and P. palmivora (b). c, d Venn diagrams show shared genes expressed in groups identified by PCA analysis for N. benthamiana (c) and P. palmivora (d). Genes with transcripts per million (TPM) ≥5 were considered to be expressed. e, f Hierarchical clustering of major classes of differentially expressed genes (p value < 10^-3, log fold change (LFC) ≥ 2) in N. benthamiana (e) and P. palmivora (f) transcriptomes. Relative expression levels of each transcript (rows) in each sample (column) are shown. TPMs were log2-transformed and then median-centred by transcript. Plant samples were centred according to the full set of mock and infected samples; only infected samples are shown. MZ axenically grown mycelium with sporangia

Fig. 4

Temporal dynamics of P. palmivora differentially expressed genes (DEGs) during infection time course. Fuzzy clustering was performed on P. palmivora DEGs. Only genes with cluster membership values ≥0.7 are shown, i.e. alpha cores (a). Functional distribution of secreted proteins for the grouped clusters is shown in (b). RXLR RXLR-effector, SCR small cysteine-rich protein, CWDE cell wall-degrading enzyme, NLP necrosis inducing protein, EPI protease inhibitor, Other other genes encoding proteins predicted to be secreted without specific functional category assigned

Fig. 5

Spatial distribution of REX effectors in N. benthamiana roots. a–d Transgenic N. benthamiana plants expressing GFP:FLAG-REX fusion proteins were regenerated from leaf explants and grown to seeds. Subcellular localisation of GFP:FLAG-REX1–4 was assessed on seedling roots stained with propidium iodide (PI). GFP:FLAG-REX1 (a), GFP:FLAG-REX2 (b) and GFP:FLAG-REX4 (d) accumulated in the cytoplasm and in the nucleus. GFP:FLAG-REX3 (c) was detected in the cytoplasm but was excluded from the nucleus. Scale bar is 10 μm

Fig. 6

REX2 and REX3 increase N. benthamiana susceptibility to P. palmivora, and REX3 interferes with host secretion. Transgenic N. benthamiana plants expressing GFP16c (control) or GFP:FLAG-REX1 to GFP:FLAG-REX4 were challenged with zoospores from P. palmivora YKDEL, and disease progression was ranked over time using the previously defined symptom extent stages (SESs). a Representative disease progression curves for transgenic plants expressing GFP:FLAG-REX1 (yellow), GFP:FLAG-REX2 (blue), GFP:FLAG-REX3 (green) or GFP:FLAG-REX4 (magenta) compared to GFP16c control plants (red dashed). p values were determined based on Scheirer-Ray-Hare nonparametric two-way analysis of variance (ANOVA) for ranked data. The experiment was carried out in duplicate (N = 22 plants). b Representative pictures of infected plants, 8 days after infection. c Disease-promoting effectors REX2 and REX3 were co-expressed with a secreted GFP construct (SP_PR1-GFP) in N. benthamiana leaves. GFP fluorescence was quantified along the nucleus

Fig. 7

The promoter of a gene encoding the secreted peptide TIPTOP is upregulated during early biotrophy in N. benthamiana roots. a Representative pictures of beta-glucuronidase (GUS)-stained whole root systems of N. benthamiana transgenics carrying TIPTOPpro::GFP:GUS, non-infected or 16 h after infection with P. palmivora LILI-tdTomato. Stars represent unstained root tips. Arrowheads represent stained root tips. b Representative pictures of infected root tips after GUS staining, showing GUS signal at the vicinity of infection sites (top panels). Uninfected root tips from the same plant do not show any staining (bottom panels). Scale bar is 25 μm. c Representative pictures of GFP signal at the root tip of infected N. benthamiana transgenics expressing GFP:GUS fusion under the control of TIPTOP promoter