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Hassan, Md Mahmudul

Publications and source records attributed to Hassan, Md Mahmudul.

4 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

A small secreted protein serves as a plant-derived effector mediating symbiosis between Populus and Laccaria bicolor

Beneficial symbiotic fungi colonize plant tissues, delivering crucial ecosystem services such as carbon sequestration and plant fertilization. Specifically, trees that form a nutrient-acquiring symbiosis with mutualistic ectomycorrhizal (ECM) fungi gain advantages from these associations by experiencing enhanced growth rates and increased resilience to both biotic and abiotic stresses. Despite the vital role ECM fungi play in the nutrition and well-being of trees, identifying key regulators participating in the molecular communication between plant and fungal cells is still in its early stages. The mutualistic relationship between Laccaria bicolor and Populus spp. has been utilized as a model system for investigating ECM symbiosis at the molecular level. It has been demonstrated that the fungus L. bicolor secretes Mycorrhiza-induced Small Secreted Proteins (MiSSPs) required for ECM development. Meanwhile, we have previously shown that P. trichocarpa small, secreted proteins (PtSSPs) are highly induced during mutualistic symbiosis and some of them can enter, via in-vitro feeding, L. bicolor hyphae affecting their growth and morphology. However, the exact role and mode of action of PtSSPs in mutualistic symbiosis remain unknown. Because previous study showed that PtSSP1 is taking up by fungal cell and then localize in fungal cells, we decide to dig further on its putative role in fungal cells and also by overexpressing it in poplar because it is not technically possible yet to overexpress poplar protein in Laccaria bicolor hyphae. Here, we further characterized the function of PtSSP1(Potri.009G063200) in ectomycorrhization, which accumulates in the nucleus of L. bicolor in an in-vitro feeding experiment. Our results provide new knowledge for the genetic engineering of plants to control associated microbes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Establishment of a genome editing tool using CRISPR-Cas9 ribonucleoprotein complexes in the non-model plant pathogen Sphaerulina musiva

CRISPR-Cas9 is a versatile genome editing system widely used since 2013 to introduce site-specific modifications into the genomes of model and non-model species. This technology is used in various applications, from gene knock-outs, knock-ins, and over-expressions to more precise changes, such as the introduction of nucleotides at a targeted locus. CRISPR-Cas9 has been demonstrated to be easy to establish in new species and highly efficient and specific compared to previous gene editing strategies such as Zinc finger nucleases and transcription activator-like effector nucleases. Grand challenges for emerging CRISPR-Cas9 tools in filamentous fungi are developing efficient transformation methods for non-model organisms. In this paper, we have leveraged the establishment of CRISPR-Cas9 genome editing tool that relies on Cas9/sgRNA ribonucleoprotein complexes (RNPs) in the model species Trichoderma reesei and developed the first protocol to efficiently transform the non-model species, Sphaerulina musiva. This fungal pathogen constitutes a real threat to the genus Populus, a foundational bioenergy crop used for biofuel production. Herein, we highlight the general considerations to design sgRNAs and their computational validation. We also describe the use of isolated protoplasts to deliver the CRISPR-Cas9 RNP components in both species and the screening for targeted genome editing events. The development of engineering tools in S. musiva can be used for studying genes involved in diverse processes such as secondary metabolism, establishment, and pathogenicity, among many others, but also for developing genetic mitigation approaches. The approach described here provides guidance for potential development of transformation systems in other non-model spore-bearing ascomycetes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Split selectable marker systems utilizing inteins facilitate gene stacking in plants

The ability to stack multiple genes in plants is of great importance in the development of crops with desirable traits but can be challenging due to limited selectable marker options. Here we establish split selectable marker systems using protein splicing elements called “inteins” for Agrobacterium-mediated co-transformation in plants. First, we show that such a split selectable marker system can be used effectively in plants to reconstitute a visible marker, RUBY, from two non-functional fragments through tobacco leaf infiltration. Next, to determine the general applicability of our split selectable marker systems, we demonstrate the utility of these systems in the model plants Arabidopsis and poplar by successfully stacking two reporters eYGFPuv and RUBY, using split Kanamycin or Hygromycin resistance markers. In conclusion, this method enables robust plant co-transformation, providing a valuable tool for the simultaneous insertion of multiple genes into both herbaceous and woody plants efficiently.

59 BASIC BIOLOGICAL SCIENCES↗