An effector-reporter system to study cellular cellular signal 1 transduction in strawberry fruit ( Fragaria ananassa )

An effector-reporter system is a powerful tool used to study cellular signal transduction; however, 12 this technique has been traditionally adopted in protoplasts. A similar system to study cellular 13 signal transduction in fruits has not yet been established. In this paper, we aim to establish an 14 effector-reporter system for strawberry fruit, a model non-climacteric fruit. 15 Results: We compared four different reporters, including GUS, GFP, FLuc, and RLuc, as well as 16 combination of two pairs of reporters, including GUS/GFP and RLuc/FLuc, by statistically 17 analyzing the pattern of reporter activities exhibited among individual fruits. Among the reporters 18 examined, GUS was the best choice. Further sampling indicated that a minimum of five mixed 19 specimens with five fruits each is necessary for an assay. With the so-established system, FaCHS 20 promoter demonstrated a sensitive response to FaMYB10 effector as well as to the external and 21 internal cues implicated in the regulation of fruit ripening. Conclusion: A step-by step protocol was established to study cellular signal transduction in 23 strawberry fruits. This finding will contribute to the molecular investigation of fruit ripening.

percentage of SEM, PSEM) to assess the variability. Specifically, according to the 122 formulas: , where SD is the standard deviation, xi is one sample 123 value; x̄ is the sample mean, and N is the sample size; and , where SE is the 124 standard error, SD is the standard deviation, and n is the number of observations of 125 the sample, PCV and PSEM were respectively determined as follows: PCV=SD/ x̄× 126 100%, and PSEM=SE/ x̄×100%. As PSEM might be caused by biological or technical 127 replicates, to understand the precise contribution of biological and technical replicates 128 to the PSEM value, we further examined the SV due to technical replicates. 129 Variability due to the technical replicates was smaller than that due to the biological 130 replicates. Meanwhile, the PSEM for GFP was much larger than that for other 131 reporters ( Table 1). As shown in Table 1 evaluated PSEM for sample size 5, and the obtained PSEM value was 10.33% (Table   152 2), which indicates that by using the mixed specimen, a sample size as low as 5 was 153 enough to satisfy the test of the significance of difference. Based on these findings, 154 we used mixed specimens with a sample size 5 for further experiments to study 155 cellular signal transduction in strawberry fruits. To characterize the effect of a ratio approach on sampling SV, we first determined the appeared to be unstable, which was reflected by high PSEM (56.45%; Table 1). This   304 Vector pCAMBIA1301, which carries GUS reporter gene driven by cauliflower 305 mosaic virus 35s promoter, was adopted to construct GUS reporter. This vector was 306 purchased from YouBio (China).

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Vector pH7WG2D.1, which carries eGFP reporter gene driven by Agrobacterium 308 rhizogenes plasmid proID promoter, was adopted to construct GFP reporter. This      14 nm for blue). GUS activity was measured using excitation set to UV mode and 377 emission at 365 nm. This activity was expressed as relative fluorescence unit (RLU).

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For GFP measurement, the protein extract was transferred into the colorimetric cup 379 and the excitation was set to blue and emission to 465 nm.  Step-by-step protocol to study cellular signal transduction 392 Step 1. Preparation of fruits 393 We used octoploid strawberry, Fragaria × ananassa Duch. (Benihoppe) in this study.

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Plants were grown in a greenhouse and fruits were divided into six developmental 395 stages as described above. Fruits at large green to white stages were chosen for the 396 study, and fruits at a uniform stage were used for each experiment. The selection of 397 fruits was based on achene color (breaking stage). Number of fruits per experiment 398 was decided as described below. Fruits were harvested by cutting from the petiole and 399 were immediately brought to the laboratory for infection.

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Step 2. Sampling design 401 Each fruit was individually infected, and five infected fruits were pooled for grinding 402 to obtain the mixed specimens. At least five biological replicates were maintained, 403 and so, for each treatment 25 fruits were maintained. For control, fruits were infected 404 with an empty vector (no effector/reporter).

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Step 3. Preparation of the bacterium carrying the target vector 406 (1) Bacterial culture 407 Liquid culture medium and antibiotic was used based on the target vector. Kanamycin