Plants respond to insect feeding with a number of defense mechanisms. Using maize genotypes derived from Antiquan germ plasm that are resistant to Lepidoptera, we have demonstrated that a unique 33-kD cysteine proteinase accumulates in the whorl in response to larval feeding. The abundance of the proteinase increased dramatically at the site of larval feeding after 1 hr of infestation and continued to accumulate for as long as 7 days. The 33-kD cysteine proteinase was most abundant in the yellow-green portion of the whorl-the normal site of larval feeding and the tissue that has the greatest inhibitory effect on larval growth in bioassays. The proteinase was expressed in response to wounding and was found in senescent leaves. It may be a marker of programmed cell death. The gene coding for the proteinase, mir1 , has been transformed into Black Mexican Sweet callus. When larvae were reared on callus expressing the proteinase, their growth was inhibited ‫ف‬ 60 to 80%. The expression of a cysteine proteinase, instead of a cysteine proteinase inhibitor, may be a novel insect defense mechanism in plants. INTRODUCTIONOver the past 25 years, maize inbreds resistant to feeding by larvae of numerous lepidopteran species have been developed from Antiguan germ plasm (Williams and Davis, 1982;Williams et al., 1990a). Inbreds derived from this germ plasm (Mp704 and Mp708) are resistant to feeding by fall armyworm ( Spodoptera frugiperda ), southwestern corn borer ( Diatraea grandiosella), European corn borer ( Ostinia nubilalis ), sugarcane borer ( D. saccharalis), tobacco budworm ( Heliothis virescens ), corn earworm ( Helicoverpa zea ), and other Lepidoptera. Fall armyworm larvae feed extensively on whorl leaf tissue, often resulting in crop losses. Genetic and quantitative trait loci analyses indicate that resistance to these Lepidoptera is a quantitative trait regulated by several genes (Williams et al., 1989;Khairallah et al., 1998). Traits such as high hemicellulose content, low protein content, and leaf toughness appear to be correlated with reduced larval growth (Williams et al., 1998). No studies have indicated conclusively that secondary products contribute to the resistance, but two-dimensional gel electrophoresis has indicated that the presence of 36-and 21-kD proteins in the whorl may be predictive of resistance (Callahan et al., 1992).Bioassays in which fall armyworm larvae are reared on lyophilized whorl tissues indicate that larvae reared on resistant material weigh ‫ف‬ 50% less than those reared on susceptible material (Williams et al., 1990b). Larvae reared on lyophilized whorl tissue from resistant genotypes are smaller, grow more slowly, and pupate later than those reared on similar material from susceptible genotypes (Chang et al., 2000). The major effect of this germplasm is to slow larval growth and development and to increase the amount of time larvae are vulnerable to predators and parasites.The same phenotype, a 50% reduction in larval growth, is apparent when larvae are reared on nonfriable callus ...

BackgroundMicrobial genome editing is a powerful tool to modify chromosome in way of deletion, insertion or replacement, which is one of the most important techniques in metabolic engineering research. The emergence of CRISPR/Cas9 technique inspires various genomic editing methods.ResultsIn this research, the goal of development of a fast and easy method for Escherichia coli genome editing with high efficiency is pursued. For this purpose, we designed modular plasmid assembly strategy, compared effects of different length of homologous arms for recombination, and tested different sets of recombinases. The final technique we developed only requires one plasmid construction and one transformation of practice to edit a genomic locus with 3 days and minimal lab work. In addition, the single temperature sensitive plasmid is easy to eliminate for another round of editing. Especially, process of the modularized editing plasmid construction only takes 4 h.ConclusionIn this study, we developed a fast and easy genome editing procedure based on CRISPR/Cas9 system that only required the work of one plasmid construction and one transformation, which allowed modification of a chromosome locus within 3 days and could be performed continuously for multiple loci.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0605-5) contains supplementary material, which is available to authorized users.

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