De Novo Design of Antimicrobial Peptides With a Special Charge Pattern and Their Application in Combating Plant Pathogens.

Plant diseases are important issues in agriculture, and the development of effective and environment-friendly means of disease control is crucial and highly desired. Antimicrobial peptides (AMPs) are known as potential alternatives to chemical pesticides because of their potent broad-spectrum antimicrobial activity and because they have no risk, or have only a low risk, of developing chemical-resistant pathogens. In this study, we designed a series of amphipathic helical peptides with different spatial distributions of positive charges and found that the peptides that had a special sequence pattern "BBHBBHHBBH" ("B" for basic residue and "H" for hydrophobic residue) displayed excellent bactericidal and fungicidal activities in a wide range of economically important plant pathogens. The peptides with higher helical propensity had lower antimicrobial activity. When we modified the peptides with a long acyl chain at their N-terminus, their plant protection effect improved. Our application of the fatty acyl-modified peptides on the leaves of tomato and Arabidopsis plants lessened the infection caused by Pectobacterium carotovorum subsp. carotovorum and Botrytis cinerea. Our study provides important insights on the development of more potent novel AMPs for plant protection.

Optogenetic Manipulation of Selective Neural Activity in Free-Moving Drosophila Adults.

Activating selected neurons elicits specific behaviors in Drosophila adults. By combining optogenetics and laser-tracking techniques, we have recently developed an automated laser-tracking and optogenetic manipulation system (ALTOMS) for studying how brain circuits orchestrate complex behaviors. The established ALTOMS can independently target three lasers (473-nm blue laser, 593.5-nm yellow laser, and 1064-nm infrared laser) on any specified body part of two freely moving flies. Triggering light-sensitive proteins in real time, the blue laser and yellow laser can respectively activate and inhibit target neurons in artificial transgenic flies. Since infrared light is invisible to flies, we use the 1064-nm laser as an aversive stimulus in operant learning without perturbing visual inputs. Herein, we provide a detailed protocol for the construction of ALTOMS and optogenetic manipulation of target neurons in Drosophila adults during social interactions.

Three-wavelength light control of freely moving Drosophila Melanogaster for less perturbation and efficient social-behavioral studies.

We developed a real-time automated laser-tracking system combined with continuous wave 1064-nm infrared or 473-nm blue lasers to provide punishment for studying memory in Drosophila Melanogaster. Combining optogenetic tools with laser properties, such as 473-nm and 593-nm lasers that activate light sensitive proteins in artificial transgenic flies, we can manipulate the specific neuron of an assigned fly among multiple flies to investigate neuron circuit relationships in social interactions. In restraining condition assay or optogenetic experiments, a ventral irradiated system would be more efficient due to higher ventral cuticle transmissions and neuron ganglia locations. Therefore, ventral irradiated systems cause less perturbation during behavior studies.

Optogenetic control of selective neural activity in multiple freely moving Drosophila adults.

We present an automated laser tracking and optogenetic manipulation system (ALTOMS) for studying social memory in fruit flies (Drosophila melanogaster). ALTOMS comprises an intelligent central control module for high-speed fly behavior analysis and feedback laser scanning (∼40 frames per second) for targeting two lasers (a 473-nm blue laser and a 593.5-nm yellow laser) independently on any specified body parts of two freely moving Drosophila adults. By using ALTOMS to monitor and compute the locations, orientations, wing postures, and relative distance between two flies in real time and using high-intensity laser irradiation as an aversive stimulus, this laser tracking system can be used for an operant conditioning assay in which a courting male quickly learns and forms a long-lasting memory to stay away from a freely moving virgin female. With the equipped lasers, channelrhodopsin-2 and/or halorhodopsin expressed in selected neurons can be triggered on the basis of interactive behaviors between two flies. Given its capacity for optogenetic manipulation to transiently and independently activate/inactivate selective neurons, ALTOMS offers opportunities to systematically map brain circuits that orchestrate specific Drosophila behaviors.