Suppressing a plant-parasitic nematode with fungivorous behavior by fungal transformation of a Bt cry gene

Abstract

BackgroundPine wilt disease, caused by the pinewood nematode Bursaphelenchus xylophilus (PWN), is an important destructive disease of pine forests worldwide. In addition to behaving as a plant-parasitic nematode that feeds on epithelial cells of pines, this pest relies on fungal associates for completing its life cycle inside pine trees. Manipulating microbial symbionts to block pest transmission has exhibited an exciting prospect in recent years; however, transforming the fungal mutualists to toxin delivery agents for suppressing PWN growth has received little attention.ResultsIn the present study, a nematicidal gene cry5Ba3, originally from a soil Bacillus thuringiensis (Bt) strain, was codon-preferred as cry5Ba3Φ and integrated into the genome of a fungus eaten by PWN, Botrytis cinerea, using Agrobacterium tumefaciens-mediated transformation. Supplementing wild-type B. cinerea extract with that from the cry5Ba3Φ transformant significantly suppressed PWN growth; moreover, the nematodes lost fitness significantly when feeding on the mycelia of the cry5Ba3Φ transformant. N-terminal deletion of Cry5Ba3Φ protein weakened the nematicidal activity more dramatically than did the C-terminal deletion, indicating that domain I (endotoxin-N) plays a more important role in its nematicidal function than domain III (endotoxin-C), which is similar to certain insecticidal Cry proteins.ConclusionsTransformation of Bt nematicidal cry genes in fungi can alter the fungivorous performance of B. xylophilus and reduce nematode fitness. This finding provides a new prospect of developing strategies for breaking the life cycle of this pest in pines and controlling pine wilt disease.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0960-5) contains supplementary material, which is available to authorized users.

Nematotoxicity of a Cyt‐like protein toxin from Conidiobolus obscurus (Entomophthoromycotina) on the pine wood nematode Bursaphelenchus xylophilus

“…Previous studies related to PFTs that target plant‐parasitic nematodes have mainly focused on Cry protein toxins found in B. thuringiensis , such as Cry5B, Cry6A, Cry1E, and Cry 55A 8,9,25 . The widely accepted mode of action of microbial disruptors of insect midgut membranes is to bind receptors and form pores on membranes, resulting in an increase in cytosolic levels of cytotoxic cations and eventual cell lysis 26 .…”

“…emamectin benzoate and abamectin), monitoring and controlling B. xylophilus vectors, and breeding of resistant trees 2,5,6 . Screening nematotoxic biological materials, such as protein toxins and plant essential oils, has been conducted to develop new strategies for B. xylophilus control 7–9 . Regarding nematocidal protein toxins, several membrane pore‐forming toxins (PFTs), such as Bacillus thuringiensis crystal proteins, have been found to be effective 8–10 …”

Engineered pine endophytic Bacillus toyonensis with nematocidal and colonization abilities for pine wilt disease control

“…Transgenic crops expressing crystal proteins have been extensively studied and applied to the control of root-knot nematodes and cyst nematodes ( Li et al, 2007 ; Kahn et al, 2021 ). The fungi that expressed crystal proteins could also reduce the population of pine wood nematodes ( Cheng et al, 2018 ). However, the techniques and strategy of pine-resistance transgenesis were not yet mature, and the breeding cycle of pine was too long.…”

“…Transgenic soybean events expressing the nematocidal protein Cry14Ab are protected from Heterodera glycines in both the greenhouse and field ( Kahn et al, 2021 ; McCarville et al, 2023 ). For PWN, the fungus Botrytis cinerea transformed with the Bt nematicidal gene Cry5Ba3 has been shown to reduce nematode fitness ( Cheng et al, 2018 ). Bacillus spp.…”

Abundance, distribution, and expression of nematicidal crystal protein genes in Bacillus thuringiensis strains from diverse habitats

“…It has been shown that some free-living nematode species are susceptible to Bt toxins (Iatsenko et al 2014 ; Wei et al 2003 ). Also, there are several studies showing that Bt Cry proteins are toxic to animal (Wei et al 2003 ; Cappello et al 2006 ; Kotze et al 2005 ; Urban et al 2013 ; Hu et al 2010 ) or to plant parasitic nematodes (Li et al 2007 , 2008 ; Guo et al 2008 ; Yu et al 2015 ; Wang et al 2012 ; Verduzco-Rosas et al 2021 ; Peng et al 2011 ; Cheng et al 2018 ; Chinheya et al 2017 ). In the toxicity studies carried out with Cry proteins against nematodes, it has been found that LC 50 values are low (summarized in Jouzani et al 2017 ), which indicates that Bt nematicidal proteins can be very useful for the control of these organisms.…”