Dissimilar Crystal Proteins Cry5Ca1 and Cry5Da1 Synergistically Act against Meloidogyne incognita and Delay Cry5Ba-Based Nematode Resistance

Abstract

Cry proteins of Bacillus thuringiensis (Bt) have been successfully used as biopesticides and in transgenic crops throughout the world. However, resources against the most serious agricultural pathogens, plant root-knot nematodes, are limited. The genomes of several highly nematicidal virulent Bt strains from our laboratory have been sequenced, facilitating the identification of novel Cry proteins and other virulence factors. We identified two novel Cry proteins, Cry5Ca1 and Cry5Da1, that exhibit high toxicity against Meloidogyne incognita. Using the Caenorhabditis elegans model, the two Cry5 toxins were shown to negatively affect nematode life span, fertility, and survival. The 50% lethal concentrations (LC 50 s) of Cry5Ca1 and Cry5Da1 were 57.22 g/ml and 36.69 g/ml, respectively. Moreover, a synergistic effect (synergism factor, 1.61 to 2.04) was observed for nematicidal toxicity of Cry5Ca1 and Cry5Da1, which is accordant with the phylogenetic results suggesting that domain II of the two novel Cry5 toxins evolved into two independent clades. Through comparison of the depressed degree of toxicity in the ␤-methylgalactoside detoxification test, we found that the novel toxin Cry5D possesses a different galactosebinding epitope; meanwhile, the finding that Cry5D does not share a motif (GXXXE) in the corresponding loop of domain II with Cry5B could explain the different galactose binding performance. Additionally, low-level cross-resistance of C. elegans bre mutant strains was evident between Cry5B and Cry5D. These results suggest that Cry5D can be used as an alternative to delay the potential resistance of nematodes to Cry5B. IMPORTANCE Although proper gene resources for Bt crops against the most serious agricultural pathogens, plant root-knot nematodes, are limited, we have identified two novel nematicidal toxins, Cry5Ca1 and Cry5Da1, against M. incognita, which have supplied more gene candidates for Bt crops designed against nematodes. Moreover, the association of the dissimilarity between Cry5Da1 and Cry5Ba1 and their low crossresistance can be attributed not only to a low sequence similarity of domain II but also to the structural difference of the key motif and receptor-binding epitope in the loops. This association facilitates the selection of a proper candidate for the prospective design of pyramided Bt crops that can delay potential resistance.KEYWORDS Bacillus thuringiensis, Caenorhabditis elegans, Cry proteins, bre resistance, nematicidal T he agricultural economic damage caused by more than 4,100 species of plantparasitic nematodes (PPNs) has been estimated to cost US$80 billion per year (1, 2), and a large proportion of this cost is due to the serious damage caused by the plant-parasitic nematode Meloidogyne incognita (3). Although many nematophagous

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

“…2 ). Other toxins, belonging to the Cry5 subfamily, such as Cry5Ca1, Cry5Da1, and Cry14 were recently found to act on PPNs, such as M. incognita and M. javanica [ 42 , 43 ]. The nematicidal cry5 subfamily genes, however, appeared to occur in low frequency in natural environments [ 44 ].…”

Mitochondria surveillance systems trigger innate immune responses to bacterial pathogens via AMPK pathway inC. elegans

“…Next we tested whether other nematicidal Bt can lead cell energy change, such as BMB171/Cry5Ca (Geng et al, 2017), BMB171/Cry6Aa (Wei et al, 2003), BMB171/Cry21Aa and non-nematicidal BMB171/Cry1Ac (Fang et al, 2009). We found that nematicidal Bt strains can cause significant energy imbalance of C. elegans comparing with non-nematicidal Bt (Fig.…”

“…To investigate the intracellular physiological changes of C. elegans after pathogenic Bt infection, we conducted the C. elegans transcriptome analysis after infection by the nematicidal Bt strain BMB171/Cry5Ba, an acrystalliferous Bt mutant BMB171 transformed with toxin gene cry5Ba on the shuttle vector pHT304 (Geng et al, 2017). As a control, we compared the transcriptome to a non-nematicidal Bt strain BMB171/pHT304, BMB171 transformed with the empty vector pHT304.…”

“…To assess whether other nematicidal Bt strains can cause MF phenomenon, we fed transgene worms SJ4143(zcIs17 [P ges-1 :: GFP mt ]) with the other four nematicidal Bt BMB171/Cry5Ca, BMB171/Cry21Aa, BMB171/Cry6Aa, and non-nematicidal Bt BMB171/Cry1Ac. These former two strains produce Cry5-like three-domain (3D) group nematicidal Cry proteins Cry5Ca (Geng et al, 2017) and Cry21Aa (Pardo-Lopez et al, 2013), respectively. While BMB171/Cry6Aa produces a non-3D group nematicidal Cry toxin Cry6Aa (Zhang et al, 2016).…”

“…Bacillus thuringiensis (Bt) is an obligate and opportunist pathogen of insect and worms, which produces insecticidal or nematicidal crystal protein during sporulation, and has been used as a leading bio-insecticide to control various pests and worms (Ruan et al, 2015). Here, we used a nematicidal Bt strain BMB171/Cry5Ba (Geng et al, 2017) and C. elegans as a model， to research the detailed mechanisms about how the cell surveillance systems sense pathogens and activate the innate immune responses. We showed that Bt infection causes mitochondrial damage, which ultimately leads to a severely cellular energy imbalance by altered of AMP/ATP ratio.…”