Transplastomic Nicotiana benthamiana plants expressing multiple defence genes encoding protease inhibitors and chitinase display broad-spectrum resistance against insects, pathogens and abiotic stresses.
Plastid engineering provides several advantages for the next generation of transgenic technology, including the convenient use of transgene stacking and the generation of high expression levels of foreign proteins. With the goal of generating transplastomic plants with multiresistance against both phytopathogens and insects, a construct containing a monocistronic patterned gene stack was transformed into Nicotiana benthamiana plastids harbouring sweet potato sporamin, taro cystatin and chitinase from Paecilomyces javanicus. Transplastomic lines were screened and characterized by Southern/Northern/Western blot analysis for the confirmation of transgene integration and respective expression level. Immunogold localization analyses confirmed the high level of accumulation proteins that were specifically expressed in leaf and root plastids. Subsequent functional bioassays confirmed that the gene stacks conferred a high level of resistance against both insects and phytopathogens. Specifically, larva of Spodoptera litura and Spodoptera exigua either died or exhibited growth retardation after ingesting transplastomic plant leaves. In addition, the inhibitory effects on both leaf spot diseases caused by Alternaria alternata and soft rot disease caused by Pectobacterium carotovorum subsp. carotovorum were markedly observed. Moreover, tolerance to abiotic stresses such as salt/osmotic stress was highly enhanced. The results confirmed that the simultaneous expression of sporamin, cystatin and chitinase conferred a broad spectrum of resistance. Conversely, the expression of single transgenes was not capable of conferring such resistance. To the best of our knowledge, this is the first study to demonstrate an efficacious stacked combination of plastid-expressed defence genes which resulted in an engineered tolerance to various abiotic and biotic stresses.
Institute of Plant Biology, College of Life Science, National Taiwan University, Taipei, Taiwan.
Peng-Jen
PJ
Chen
Rajendran
R
Senthilkumar
Wann-Neng
WN
Jane
Yong
Y
He
Zhihong
Z
Tian
Kai-Wun
KW
Yeh
D000528:Alternaria / Q000187:drug effects / Q000502:physiology; D000818:Animals; D001681:Biological Assay; D002688:Chitinases / Q000235:genetics*; D003433:Crosses, Genetic; D060467:Disease Resistance / Q000187:drug effects / Q000235:genetics* / Q000276:immunology; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D017343:Genes, Plant; D005822:Genetic Vectors / Q000378:metabolism; D060434:Herbivory / Q000187:drug effects; D007313:Insecta / Q000502:physiology*; D008563:Membrane Lipids / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D010269:Paraquat / Q000494:pharmacology; D016973:Pectobacterium carotovorum / Q000187:drug effects / Q000502:physiology; D010935:Plant Diseases / Q000235:genetics / Q000276:immunology / Q000382:microbiology; D018515:Plant Leaves / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D030821:Plants, Genetically Modified; D018087:Plastids / Q000187:drug effects / Q000235:genetics* / Q000648:ultrastructure; D011480:Protease Inhibitors / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013312:Stress, Physiological / Q000187:drug effects / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics* / Q000276:immunology / Q000382:microbiology* / Q000469:parasitology; D014170:Transformation, Genetic / Q000187:drug effects
D008563:Membrane Lipids; D011480:Protease Inhibitors; D012965:Sodium Chloride; D002688:Chitinases; D010269:Paraquat
CeCPI; chitinase; gene stacking; plastid transformation; sporamin; stress tolerance