Preventive Effects of Fluoro-Substituted Benzothiadiazole Derivatives and Chitosan Oligosaccharide against the Rice Seedling Blight Induced by Fusarium oxysporum

Rice seedling blight, caused by Fusarium oxysporum, significantly affects global rice production levels. Fluoro-substituted benzothiadiazole derivatives (FBT) and chitosan oligosaccharide (COS) are elicitors that can enhance plant resistance to pathogen infection. However, there is a lack of information regarding FBT and COS used as elicitors in rice seedlings blight. Therefore, the aim of this study was to evaluate the effect of FBT and COS treatments on rice seedling blight and elucidate the molecular mechanisms of the two elicitors for inducing resistance using proteomic technique. Results indicated that FBT and COS significantly reduced the disease incidence and index, and relived the root growth inhibition caused by F. oxysporum (p < 0.05). Biochemical analyses demonstrated that these two elicitors effectively enhanced activities of defense enzymes. Moreover, the proteomic results of rice root tissues disclosed more differentially expressed proteins in diterpenoid biosynthesis pathway that were particularly stimulated by two elicitors compared to the other pathways studied, resulting in the accumulation of antimicrobial substance, momilactone. Findings of this study could provide sound theoretical basis for further applications of FBT and COS used as rice elicitors against seedling blight.


Benzothiadiazole Derivatives and Chitosan
Oligosaccharide against the Rice Seedling Blight

Induced by Fusarium oxysporum
The Detailed Description of Sample Preparation for Proteomics

Trypsin Digestion
For digestion, the protein solution was reduced with 10 mM DTT for 1 h at 37 °C and alkylated with 20 mM IAA for 45 min at room temperature in darkness. For trypsin digestion, the protein sample was diluted by adding 100 mM TEAB to urea concentration less than 2M. Finally, trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratio for a second 4 h-digestion. Approximately 100 µg protein for each sample was digested with trypsin for the following experiments.

TMT Labeling
After trypsin digestion, peptide was desalted by Strata X C18 SPE column (Phenomenex) and vacuum-dried. Peptide was reconstituted in 0.5 M TEAB and processed according to the manufacturer's protocol for 6-plex TMT kit. Briefly, one unit of TMT reagent (defined as the amount of reagent required to label 100 µg of protein) were thawed and reconstituted in 24 µl ACN. The peptide mixtures were then incubated for 2 h at room temperature and pooled, desalted and dried by vacuum centrifugation.

HPLC Fractionation
The sample was then fractionated into fractions by high pH reverse-phase HPLC using Agilent 300 Extend C18 column (5 µm particles, 4.6 mm ID, 250 mm length). Briefly, peptides were first separated with a gradient of 2% to 60% acetonitrile in 10 mM ammonium bicarbonate pH 10 over 80 min into 80 fractions. Then, the peptides were combined into 18 fractions and dried by vacuum centrifuging.
The gradient was comprised of an increase from 6% to 22% solvent B (0.1% FA in 98% ACN) over 22 min, 22% to 36% in 10 min and climbing to 85% in 5 min then holding at 85% for the last 3 min, all at a constant flow rate of 400 nl/min on an EASY-nLC 1000 UPLC system, The resulting peptides were analyzed by Q ExactiveTM plus hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific).
The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM plus (Thermo) coupled online to the UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using NCE setting as 30; ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 1E4 in the MS survey scan with 30.0s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the orbitrap; 5E4 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan range was 350 to 1800. Fixed first mass was set as 100 m/z.    Figure S1 Illustration of KEGG pathway of terpenoid backbone biosynthesis in oligochitosan-treated roots. Red means up-regulated proteins in FBT and COS-treated roots compared to control root.