Agrobacterium tumefaciens-mediated transformation and expression of GFP in Ascochyta lentis to characterize ascochyta blight disease progression in lentil

Abstract

The plant immune system is made up of a complex response network that involves several lines of defense to fight invading pathogens. Fungal plant pathogens on the other hand, have evolved a range of ways to infect their host. The interaction between Ascochyta lentis and two lentil genotypes was explored to investigate the progression of ascochyta blight (AB) in lentils. In this study, we developed an Agrobacterium tumefaciens-mediated transformation system for A. lentis by constructing a new binary vector, pATMT-GpdGFP, for the constitutive expression of green fluorescent protein (EGFP). Green fluorescence was used as a highly efficient vital marker to study the developmental changes in A. lentis during AB disease progression on the susceptible and resistant lentil accessions, ILL6002 and ILL7537, respectively. The initial infection stages were similar in both the resistant and susceptible accessions where A. lentis uses infection structures such as germ tubes and appressoria to gain entry into the host while the host uses defense mechanisms to prevent pathogen entry. Penetration was observed at the junctions between neighbouring epidermal cells and occasionally, through the stomata. The pathogen attempted to penetrate and colonize ILL7537, but further fungal advancement appeared to be halted, and A. lentis did not enter the mesophyll. Successful entry and colonization of ILL6002 coincided with structural changes in A. lentis and the onset of necrotic lesions 5-7 days post inoculation. Once inside the leaf, A. lentis continued to grow, colonizing all parts of the leaf followed by plant cell collapse. Pycnidia-bearing spores appeared 14 days post inoculation, which marks the completion of the infection cycle. The use of fluorescent proteins in plant pathogenic fungi together with confocal laser scanning microscopy, provide a valuable tool to study the intracellular dynamics, colonization strategy and infection mechanisms during plant-pathogen interaction.

Fig 1

(A) Schematic diagram of tandem T-DNA sequences at the insertion site in AlKewell82-GFP prepared by SnapGene Viewer (GSL Biotech; available at snapgene.com) (B) PCR detection of the T-DNA and flanking host sequences using primers JD425 and JD381 (917 bp) and JD321 and JD427 (707 bp) for bands 1 and 2, respectively, and indicated in Fig 1A.

Fig 2. Characterization of AlKewell82-GFP transformant.

Radial growth and sporulation of (A) untransformed AlKewell and (B) AlKewell82-GFP. Sporulation of (C) AlKewell and (D) AlKewell82-GFP on ILL6002. (E) Pathogenicity of AlKewell and AlKewell82-GFP on ILL6002 and ILL7537.

Fig 3

Fungal development during early stages of infection (A) Germination of conidia on the leaf cuticle at one 1 DPI (B) trichomes at 1 DPI (C) Formation of an appressorium (ap) at one DPI; (D) AlKewell82-GFP mycelia after five DPI. Scale bars are 50 μm, inset: scale bar is 10 μm.

Fig 4. Penetration structures and early colonization events.

(A) Unlobed hyphae (h) at one DPI (B) Propidium iodide-stained ILL6002 infected with AlKewell82-GFP. ROS production by ILL6002 in response to (C) AlKewell and (D) AlKewell82-GFP at one DPI (E) Surface growth of AlAlKewell82-GFP hyphae and stomatal entry (s). (F) 3D representation of (E) with stomatal cavity indicated (arrow). All scale bars are 50 μm.

Fig 5. Cellular reaction of susceptible and resistant varieties to AlKewell82-GFP during early, mid and late phase.

Inset in ILL6002 mid-infection phase, magnified image of enlarged hyphae localised within the leaf. Scale bars are 50 μm.

Fig 6

Observations of AlKewell82-GFP at 7 DPI on ILL6002 (A) and ILL7537 (B) using confocal microscopy and optical sections at the leaf surface (i) and (ii) for each host genotype. Inset images are Z-projections of optical sections of the magnified areas indicated by the square on the main image (dotted line). For ILL6002, the leaf surface (A.i) and mesophyll (A.ii) sections were 11.2 μm and 12.8 μm in thickness and separated by 12.8 μm distance. For ILL7537, the leaf surface (B.i) and mesophyll (B.ii) sections were 4.8 μm and 9.6 μm in thickness, respectively, and separated by 4.0 μm distance. All scale bars are 50 μm.