While select plants have been utilized for metabolic engineering, individual species have undergone limited host engineering. This highlights a current need for the field, as host engineering could be a valuable means to improve the yields and quality of plant-produced biomolecules. Plants are increasingly used for the production of antibodies; however, their use has been hindered due to differences in N-glycosylation patterns compared to mammalian cells . Strasser et al. utilized host engineering to ameliorate this issue. Using RNAi, genes encoding a ß-1,2-xylosyltransferase and an α-1,3-fucosyltransferase were knocked down in N. benthamiana. This resulted in a strain of N. benthamiana capable of producing the antibody 2G12 with the same glycosylation as 2G12 produced in Chinese hamster ovary cells without noticeable effects on plant health . Strains of soybean and Arabidopsis have also been engineered with the use of a feedback-insensitive variant of cystathionine gamma synthase, which leads to the accumulation of methionine . These strains can serve as a platform for further engineering of nutraceuticals such as the methionine-derived glucosinolate, glucoraphanin. Additionally, hydroponic nft system improving the biomass of production platforms could further improve yields. One study generated tobacco plants expressing a synthetic glycolate pathway that improved biomass productivity by 19–37% .
The use of this strain could improve the viability of plants as a production platform for therapeutic small molecules. Host engineering could prove to be a powerful tool for plant synthetic biologists to push the boundaries of their production platform.Fruit, nut, and berry crops are commonly grouped into one of three categories: temperate, subtropical, and tropical. Temperate zone crops include almond, apple, apricot, peach, grape, blueberry, and strawberry . Avocado, citrus, and guava are considered to be subtropical, while banana, cashew, and pineapple are tropical . Generally, temperate and subtropical crops can be grown in San Mateo and San Francisco Counties , but tropical crops are rarely successful. This publication focuses on temperate and subtropical crops. Temperate zone crops generally require a period of cold temperature during the winter months for successful flower and fruit development. This cold temperature period is measured in “chill hours” . Some crops require many chill hours, while others require few. This is called the crop’s “chill requirement.” When selecting temperate zone crops, it is important to choose only those crops that have a chill requirement that will be met at your location. Subtropical crops, such as citrus, loquat, and guava, require little or no chilling. Native to warm-climate regions, these crops can be injured by cold temperatures during winter and spring months, and they require heat during the growing season for fruit maturation and flavor.Selecting climate zones and meeting chill requirements are not the only factors necessary for good fruit production. Pollination, sunlight, heat accumulation, and wind are all important considerations.
For fruit development to occur, flowers have to be pollinated; that is, pollen must move from the male organs to the female organs . Pollen transfer can be facilitated by bees, beetles, flies, butterflies, moths, birds, bats, wind, and water. The pollen can come from flowers on the same tree , or from flowers of other trees of the same species . In some cases, a crop may require another tree of a specific variety for pollination. For information on pollination requirements, see the “Crop and Variety Selection Table” at the end of this publication. For further details, see “Notes on Crops and Varieties” below. Note that even in self fertile crops, cross-pollination can increase fruit set. Also, poor pollination can occur as a result of insufficient pollinator activity, such as during cool and wet weather in the spring when bees may be less active . For most crops, plentiful sunlight and warm temperatures during the growing season are needed. Factors such as fog, wind, elevation, aspect , and distance from the ocean affect sunlight level and temperature.For some crops, wind protection will be needed. Leaf burn, leaf drop, crown deformation, fruit drop, and fruit scarring can occur in locations exposed to wind, such as near the coast or along hilltops and ridges. Generally, when choosing sites for temperate zone crops, select locations where it is warmest in the summer, coldest in the winter, and most protected from wind and salt spray. For subtropical crops, select locations where it is warmest in the summer, mildest in the winter, and protected from wind and salt spray.Mosquitoes rely on the olfactory system to find plants as a source of carbohydrates, hosts for blood meals, and oviposition sites. Because pathogens might be transmitted during a bite by an infected mosquito, there is understandably a great deal of interest in unraveling the olfactory aspects of human-mosquito interactions to explore ways of reducing mosquito bites.
However, plant nectar sources are often essential for mosquitoes because they increase mosquito life span and reproductive capacity and long-living mosquitoes are more dangerous . Therefore, understanding how mosquitoes find plants/flowers is also important for reducing the transmission of vector-borne diseases. Previously, we have identified generic and plant kairomone sensitive odorant receptors from the Southern house mosquito, Culex quinquefasciatus . One of these ORs, CquiOR1, belongs to a cluster of 6 ORs from the Southern house mosquito and 2 ORs from the yellow fever mosquito, Aedes aegypti . Specifically, CquiOR2, CquiOR4, AaegOR14, AaegOR15, CquiOR5, CquiOR84, and CquiOR85. Of note, CquiOR2 is not the previously reported oviposition attractant-detecting OR2 , which has been renamed CquiOR121 . We cloned CquiOR2 and the other ORs in this cluster . We then deorphanized these receptors using the Xenopus oocyte recording system and a panel of odorants with physiologically and behaviorally relevant compounds, including oviposition attractants, mosquito repellents, and plant-derived compounds . Here, we report that these receptors, particularly CquiOR4, CquiOR5, and AaegOR15, are very sensitive to plant-derived compounds, including repellents. CquiOR4, for example, which is very specific to female antennae, with high and low transcript levels in nonblood fed and blood-fed mosquitoes, respectively, showed a robust response to the natural repellent 2-phenylethanol. Repellency activity elicited by 2-phenylethanol reduced significantly in CquiOR4-dsRNA-treated mosquitoes, but it was unchanged when these mosquitoes were tested against DEET, which is detected with another receptor .The bioassay arena was modified from our surface-landing assay initially designed to mimic a human arm without odors or humidity. CO2 at 50 mL/min was added to activate female mosquitoes, and blood was provided as both an attractant and a reward. In short, two 50-mL Dudley bubbling tubes, painted internally with a black hobby and craft enamel , were held in a wooden board , 17 cm apart from each end and 15 cm from the bottom. The board was attached to the frame of an aluminum collapsible field cage . Two small openings were made 1 cm above each Dudley tube to hold two syringe needles to deliver CO2. To minimize the handling of mosquitoes, test females were kept inside collapsible field cages since the latest pupal stage. These female cages had their cover modified for behavioral studies. A red cardstock was placed internally at one face of the cage, and openings were made in the cardboard and cage cover so the cage could be attached to the wooden board with the two Dudley tubes and CO2 needles projecting inside the mosquito cage 6 and 3 cm, respectively. Additionally, windows were made on the top and the opposite end of the red cardstock for manipulations during the assays and a video camera connection, respectively. The mosquito cage housing 30–50 test females was connected to the platform holding the Dudley tubes at least 2h before bioassays. At least 10 min before the assays, water at 38 °C started to be circulated with a Lauda’s Ecoline water bath, nft channel and CO2 at 50 mL/min was delivered from a gas tank just at the time of the behavioral observations. Sample rings were prepared from strips of filter papers 25 cm long and 4 cm wide and hung on the cardstock wall by insect pins to make a circle around the Dudley tubes. Cotton rolls were loaded with 100 μL of defibrinated sheep blood purchased from the University of California, Davis, VetMed shop and placed between a Dudley tube and a CO2 needle. For each run one paper ring was loaded with 200 μL of hexane and the other with 200 μL tested compounds at a certain concentration in hexane. The solvent was evaporated for 1–2 min, blood-impregnated cotton plugs, and filter paper rings were placed in the arena, CO2 was started, and the assays were recorded with a camcorder equipped with Super NightShot Plus infrared system .
During the assay, the arena was inspected with a flashlight whose lens was covered with a red filter. After 5 min, the number of females that landed and continued to feed on each side of the arena was recorded. Insects were gently removed from the cotton rolls, and the assays were reinitiated after rotation of sample and control. Thus, repellency for each set of test mosquitoes was measured with the filter paper impregnated with the same sample at least once on the left and once on the right side of the arena. After three runs, filter paper strips and cotton plugs were disposed of, and new loads were prepared.We cloned the cDNAs for CquiOR2, CquiOR4, CquiOR5, CquiOR84, CquiOR85, AaegOR14, and AaegOR15, which are clustered along CquiOR1 . Then, each OR was separately coexpressed along with its coreceptor in Xenopus oocytes for deorphanization. We used a panel of odorants containing oviposition attractants, repellents, plant-derived compounds and other physiologically or behaviorally relevant compounds. We expected that the odorant profiles for these receptors would resemble that of CquiOR1 , but found marked differences. For example, three floral compounds elicited robust currents on CquiOR4/CquiOrco-expressing oocytes. Specifically, these oocytes were very sensitive to 2-phenylethanol, phenethyl formate, and, propionate . To obtain more information about the sensitivity of the CquiOR4 + CquiOrco receptor, we performed concentration-response analyses for the three best ligands. Currents elicited by 2- phenylethanol were already saturated at the normal screening dose of 1 mM. Thus, weperformed these analyses with concentrations in the range of 0.1 μM to 0.1 mM . 2- Phenylethanol was indeed the most potent of the compounds in our panel, activating CquiOR4 + CquiOrco with EC50 of 28 nM. CquiOR5 + CquiOrco receptor showed a quite different profile, with the three best ligands being linalool, p-methane-3,8-diol and linalool oxide . Although the responses were not as robust as those elicited by the best ligands for CquiOR4 + CquiOrco, they were dose-dependent . Linalool activated CquiOR5/CquiOrco-expressing oocytes with an EC50 of 574 nM. By contrast, no compounds in our panel elicited relevant currents in CquiOR2/CquiOrco-expressing oocytes, except for isopentyl acetate . On the other hand, CquiOR84/CquiOrco-expressing oocytes responded with large currents when challenged with N–3-methyl-benzamide or PMD . Similar responses were recorded with CquiOR85 + CquiOrco receptor. The two receptors from Ae. aegypti in the cluster, ie, AaegOR14 and AaegOR15, gave remarkably different responses in terms of profile and sensitivity. AaegOR14/AaegOrcoexpression oocytes elicited only weak currents when challenged with 4-methylphenol and 4- ethylphenol . By contrast, AaegOR15/AaegOrco-expressing oocytes generated dose-dependent, robust currents when challenged with phenethyl propionate, phenethyl formate, and acetophenone . As far as the two major ligands are concerned, AaegOR15 profile resembles that of CquiOR4 + CquiOrco. 3.2. Tissue expression analysis The best ligands for CquiOR4 and CquiOR5 have been previously reported to have repellency activity, particularly the commercially available PMD and 2-phenylethanol . To get a better insight into the possible role of these receptors in repellency behavior, we performed qPCR analyses. We surmised that transcript levels of the receptor for repellents and host attractants would decrease after a blood meal because blood-fed mosquitoes cease host finding. By contrast, transcripts of receptors involved in the reception of oviposition attractants would increase given that gravid females use their olfactory system to locate suitable sites for oviposition. Additionally, this receptor was highly enriched in female antennae, with only basal levels in maxillary palps, proboscis, and legs thus further supporting a possible role in hosting finding or repellency activity. By contrast, transcript levels of CquiOR5 did not change significantly after a blood meal . Moreover, this receptor is not specific to or enriched in female antennae. We found high transcript levels in the proboscis, maxillary palps, and legs .We performed RNAi experiments to test whether reducing transcript levels of CquiOR4 would affect repellency activity. Similarly, we tested CquiOR5 vis-à-vis the best ligands. First, we compared the transcript levels of these genes in water-, β-galactosidase-dsRNA and CquiOR4-dsRNA-injected mosquitoes.