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.

Targeted Disruption of Scytalone Dehydratase Gene Using Agrobacterium tumefaciens-Mediated Transformation Leads to Altered Melanin Production in Ascochyta lentis

“…Ascochyta lentis Al Kewell, obtained from Kewell, Victoria [ 15 , 18 , 19 ] was grown in half strength potato dextrose agar (PDA, BD Difco, Sparks, MD, USA) supplemented with 30 µg mL −1 streptomycin (Astral Scientific, Gymea, Australia) at room temperature under a 12 h fluorescent light regime to induce sporulation. Maintenance and spore preparation of A. lentis isolates were performed as described by Davidson et al (2016) [ 15 ].…”

“…The lentil accessions used in this study include the susceptible control ILL6002, resistant control ILL7537 together with Australian cultivars PBA Bolt, PBA Hurricane XT and Nipper. Lentil accessions PBA Hurricane XT and Nipper have been previously described [ 15 , 18 , 20 ] as susceptible and resistant to Al Kewell, respectively. Lentil varieties were obtained from Seednet (Horsham, Australia) and Pulse Breeding Australia (Horsham, Australia).…”

“…Lentil varieties were obtained from Seednet (Horsham, Australia) and Pulse Breeding Australia (Horsham, Australia). Plants were grown as described by Henares et al (2019) [ 18 ].…”

“…To create a vector for targeted gene replacement via ATMT, we modified pATMT-GpdGFP [ 18 ] by removing its GFP expression cassette and inserting two Kpn I (NEB, Ipswich, MA, USA) restriction sites between the Pt rp C promoter of the hygromycin B cassette and the left border sequence and between the T trp C terminator and the right border sequence, respectively. To achieve this, the plasmid backbone was PCR amplified from left to right border using primers JD423 and JD424 ( Table 1 , Figure S1 ).…”

“…The mechanism by which A. lentis infects lentil is still poorly understood, in part due to the limited biochemical tools available to study gene function in this pathogen. Recently, an Agrobacterium tumefaciens based transformation has been reported for A. lentis [ 18 ]; however, targeted gene manipulation has not been described for this pathogen.…”