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.
Highlights
Lentil (Lens culinaris Medik.) is one of the earliest domesticated crops and is sometimes referred to as one of the ancient grains in modern times [1]
The resulting T-DNA was flanked by left and right borders and contained the hygromycin selectable marker with the hygromycin B phosphotransferase resistance gene driven by the Aspergillus nidulans tryptophan C promoter (PtrpC), and the reporter gene egfp under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter (PgpdA) (S1 Fig) The plasmid sequence of pATMT-GpdGFP can be found in the Supporting Information (S1 and S2 Files)
To help visualize and fully investigate the initiation and development of ascochyta disease in lentil, a fluorescent strain of A. lentis was produced using protocols developed for the genetic transformation of Aspergillus awamori [26]
Summary
Lentil (Lens culinaris Medik.) is one of the earliest domesticated crops and is sometimes referred to as one of the ancient grains in modern times [1]. This high-value crop is an excellent source of high quality plant protein for human nutrition. The Australian lentil industry produces an average of 292,000 tonnes annually and production is centred in the Southern temperate cropping zones of South Australia and Victoria [3]. Average annual yield and grain quality losses in Australia amount to A$0.9 M and control costs add a further A$15.3 M penalty to the Australian lentil industry [7]
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