Visualization of Phytophthora palmivora Infection in Oil Palm Leaflets with Fluorescent Proteins and Cell Viability Markers

Abstract

Bud rot (BR) is the most devastating disease affecting oil palm (Elaeis guineensis) crops in Colombia. Its causal agent, Phytophthora palmivora, initiates the infection in immature oil palm leaflets producing necrotic lesions, followed by colonization of opportunistic necrotrophs, which increases disease damage. To improve the characterization of the disease, we transformed P. palmivora using Agrobacterium tumefaciens-mediated transformation (ATMT) to include the fluorescent proteins CFP-SKL (peroxisomal localization), eGFP and mRFP1 (cytoplasmic localization). The stability of some transformants was confirmed by Southern blot analysis and single zoospore cultures; additionally, virulence and in vitro growth were compared to the wild-type isolate to select transformants with the greatest resemblance to the WT isolate. GFP-tagged P. palmivora was useful to identify all of the infective structures that are commonly formed by hemibiotrophic oomycetes, including apoplastic colonization and haustorium formation. Finally, we detected cell death responses associated with immature oil palm tissues that showed reduced susceptibility to P. palmivora infection, indicating that these tissues could exhibit age-related resistance. The aim of this research is to improve the characterization of the initial disease stages and generate cell biology tools that may be useful for developing methodologies for early identification of oil palm materials resistant or susceptible to BR.

Fig. 2. Fluorescence distribution observed in colonies transformed with eGFP, mRFP1 and CFP-SKL. Microscope slides were mounted with mycelia from a 5-day-old culture grown on 20%-V8 plates. The scale bar represents 25 μm.

Fig. 3. GFP-labeled P. palmivora during colonization at different times in oil palm immature leaflets. A: Appressorium, GC: Germinating cyst, AH: Apoplastic hyphae, HS: Hyphal swelling, H: Haustorium, Sp: Sporangium. The scale bar represents 50 μm.

Fig. 4. Necrotic lessons observed on immature leaflets inoculated with a WT and GFP-tagged P. palmivora. (A) Soft tissue from minus five leaf and rigid immature tissue from minus three leaf inoculated with P. palmivora, observed at four days post-inoculation. (B) Wounded or unwounded green spear leaflets four days after inoculation with P. palmivora. The red arrow shows the site that were wounded with a sterile syringe needle.

Fig. 5. Cell death responses observed in distal and rigid parts of immature leaflets inoculated with a GFP-tagged transformant and counterstained with PI. (A) Proximal soft tissue inoculated at 24 hpi. (B) Germinating cysts of P. palmivora inoculated in a distal part of an immature leaflet at two hpi. (C), (D) Oil palm cell death responses in a rigid immature leaflet at 12 (C) and 48 (D) hpi, respectively. The scale bars represent 100 μm. A: Appressorium, GC: Germinating cyst, PpN: P. palmivora cell nucleus, EgN: Oil palm (E. guineensis) cell nucleus.

Fig. 1. (A) Schematic representation of the T-DNA included in the binary plasmid pNPTII. FP: Fluorescent protein. (B) Representative evaluation of transformant colonies by Southern blot with nptII probe three months after transformation; a wild type (WT) isolate was used as a negative control. Dashed lines show the colonies selected for monosporic cultures. The bottom blot shows the hybridization obtained from colonies derived from different single zoospore cultures.

Table 1. List of primers used in this study

Table 2. Number of transformant colonies obtained, discriminated by isolate, fluorescent protein and copy number determined by South-ern blot

Table 3. Comparisons of colony diameter and lesson length in oil palm leaflets of different transformants