Table 1.
Primers used in this study.
Fig 1.
Structure of Heterodera avenae FAR protein genes Ha-far-1 and Ha-far-2.
The structures of Ha-far-1 and Ha-far-2 were determined by genomic DNA to cDNAs from start codon to stop codon. Exon (rectangles), introns (solid lines) and intron phase (0, 1, 2) were shown.
Fig 2.
Phylogenetic tree containing FAR proteins of nematodes.
A neighbor-joining phylogenetic tree with protein sequences of Ha-FAR-1, Ha-FAR-2, Hf-FAR-1 (marked with asterisks) and the 24 amino acid sequences of FAR proteins from free-living, animal-parasitic and plant-parasitic nematodes. The phylogenetic tree was constructed by Mega 6.0 after aligning the protein sequences with ClustalW. The percentage bootstrap values were inferred from 1,000 replicates and are indicated at the nodes. Scale bar represents 0.2 amino acid substitutions per site.
Fig 3.
Secondary structure of cereal cyst nematode FAR proteins.
Ha-FAR-1,Ha-FAR-2 and Hf-FAR-1 contained a putative signal peptide at the N terminus (long dark blue rod) and were rich in alpha helix (long vertical blue lines) and random coil (short vertical violet lines) structures. An extended strand (moderate vertical red lines) was detected in Ha-FAR-2. Ha-FAR-1 had two predicted casein kinase II phosphorylation sites at amino acids 71 and 81, and Hf-FAR-1 has one at amino acid 71 (red arrow). NetNGlyc 1.0 Server indicated that neither Ha-FAR-1 nor Hf-FAR-1 has a predicted N-glycosylation site but that Ha-FAR-2 contains predicted N-glycosylation sites (double sided blue arrows) at amino acids 32, 84 and 94.
Fig 4.
Detection of recombinant proteins of Ha-FAR-1, Ha-FAR-2 and Hf-FAR-1 via SDS-PAGE
M: Protein marker; 1, 3, 5: Supernatant of unpurified Hf-FAR-1, Ha-FAR-1, Ha-FAR-2 respectively; 2, 4, 6: Purified Hf-FAR-1, Ha-FAR-1, Ha-FAR-2 respectively.
Fig 5.
Diverse binding activates to DAUDA of Ha-FAR-1,Ha-FAR-2 and Hf-FAR-1.
A: Fluorescence emission spectra (excitation at 345 nm) of 3μM DAUDA in buffer alone or with buffer plus, 3μM Ha-FAR-1 complex, 3μM Ha-FAR-2 complex or 3μM Hf-FAR-1 complex. B, C, D: Titration curves for determining the dissociation constant (Kd) for interaction of DAUDA with Ha-FAR-2, Hf-FAR-1 and Ha-FAR-1.
Fig 6.
Diverse binding abilities to retinol of Ha-FAR-1,Ha-FAR-2 and Hf-FAR-1.
A: Fluorescence emission spectra (excitation at 350 nm) of 10μM retinol in buffer alone or in buffer plus 2.5μM Ha-FAR-1 complex, 2.5μM Ha-FAR-2 complex or 2.5μM Hf-FAR-1 complex. Peak emission was at 470nm. B, C, D: Titration curves for determining the dissociation constant (Kd) for interaction of retinol with Ha-FAR-2, Hf-FAR-1 and Ha-FAR-1.
Fig 7.
Competition analysis of oleic acid-displaced DAUDA (A) or retinol (B) from Ha-FAR-1 binding sites. A: The reversed change of fluorescence intensity (excitation at 345 nm) was observed after the addition of oleic acid to the 10μM DAUDA +1.5μM Ha-FAR-1 complex. The wavelengths of peak emission by DAUDA were changed from 499 nm to 530 nm. B: Fluorescence intensity (excitation at 350 nm) produced a pronounced drop after adding oleic acid to 10μM retinol +2.5μM Hf-FAR-1 complex.
Fig 8.
Localization of Ha-far-1 and Ha-far-2 in pre-J2 of H. avenae by in situ hybridization.
Hybridization to antisense Ha-far-1 (A) or Ha-far-2 (C) by a DIG-labeled cDNA probe showed that the Ha-far-1and Ha-far-2 are both located in the hypodermis. The hybridization with DIG- labeled sense cDNA probe of Ha-far-1 (B) or Ha-far-2 (D) was used as control.
Fig 9.
Relative expression of Ha-far-1 and Ha-far-2 in five developmental stages was determined using RT-qPCR.
The relative expression was calculated with 2-∆∆Ct method by normalizing with two internal reference β-actin gene and GAPDH gene and presented as the change in mRNA level in various nematode developmental stages relative to that of Ha-far-2 at preJ2. preJ2: pre-parasitic second-stage juvenile; J2, J3 and J4: second-, third- and fourth-stage juvenile, respectively; F: female.