Fig 1.
Multiple alignment of annexins from Heterodera avenae and some other plant-parasitic nematodes.
In the consensus, residues in the black background are totally identical; boxed areas are four conserved annexin domains predicted by the NCBI Conserved Domain Search Service; signal peptides of H. schachtii and H. glycines analyzed by Signal P 4.0 are underlined. SP, signal peptide; Gp, Globodera pallida; Bx, Bursaphelenchus xylophilus; Hs, H. schachtii; Hg, H. glycines.
Fig 2.
Southern blot, in situ hybridization and developmental expression pattern analysis of Ha-annexin.
(A) Southern blot analysis of Ha-annexin. Genomic DNA from H. avenae was digested with BamHI and EcoRI, respectively, and probed with the Dig-labeled CDS of Ha-annexin. They had 3 and 2 signal strips, respectively. (B) In situ hybridization of the Ha-annexin transcripts in pre-parasitic second-stage juveniles. Signal of antisense Ha-annexin DIG-Labeled cDNA probes localized within the subventral glands (SVGs), with sense probes as a negative control. The SVG, metacorpus (M), and stylet (S) are indicated with arrows. Scale bar = 20 μm. (C) Developmental expression pattern of Ha-annexin. The relative expression level of Ha-annexin was quantified using qPCR for six different H. avenae stages. The fold change values were calculated using the 2-ΔΔCt method and presented as the change in mRNA level in various nematode developmental stages relative to that of egg. Each column represents the mean of 3 independent assays with standard deviation. preJ2: pre-parasitic second-stage juvenile; parJ2, J3 and J4: parasitic second-, third- and fourth-stage juvenile, respectively.
Fig 3.
Subcellular localization of Ha-ANNEXIN in the plant cell.
(A) pUC35S:ANNEXIN:GFP fusion construct and pUC35S:GFP control construct were transformed into onion epidermal cells by bombardment. Scale bar = 100 μm. (B) Agrobacterium tumefaciens cells carrying pCamv35S:ANNEXIN:GFP fusion and pCamv35S:GFP were transiently expressed in Nicotiana benthamiana cells. Scale bar = 20 μm. Western blotting of N. benthamiana leaves infiltrated with pCamv35SGFP-annexin showed expected size of annexin-GFP fusion, which is larger than GFP control. In both (A) and (B), GFP signals were observed in the whole transformed cells for annexin-GFP fusion, which is the same as GFP control.
Fig 4.
Effect of BSMV-HIGS of Ha-annexin on infection of wheat roots by H. avenae.
(A) At 7 dpi, the expression of Ha-annexin in nematodes recovered from wheat inoculated by BSMV:annexin was not detected by qPCR with BSMV:00 and BSMV:eGFP as positive controls. Nematode infection of wheat inoculated by BSMV:annexin compared to the blank negative control BSMV:00 and the negative control BSMV:eGFP showed a highly significant reduction in the number of juveniles/plant at 7 dpi (B) and females/plant at 40 dpi (C) by Duncan test (P<0.01). Each column represents the mean with standard deviation.
Fig 5.
Suppression of BT-PCD by Ha-ANNEXIN.
(A) Assay for the suppression of BAX-triggered cell death (BT-PCD) in Nicotiana benthamiana by Ha-ANNEXIN. N. benthamiana leaves were infiltrated with buffer or Agrobacterium tumefaciens cells containing a vector carrying the Ha-annexin gene or the negative control eGFP gene, either alone or followed 24 h later with A. tumefaciens cells carrying a mouse Bax gene. Photos of phenotypes of infiltrated leaves of N. benthamiana were taken approximately 7 days after the last infiltration. Results of the verification of gene expression of Ha-annexin, eGFP and Bax by RT-PCR or western blotting are shown below. (B) Necrosis Index of Ha-ANNEXIN and control eGFP followed by BAX. Each column represents the mean with standard deviation. The column with asterisks indicate a highly statistically significant reduction of the Necrosis Index of Ha-ANNEXIN compared with that of eGFP by t-test (P<0.01).
Fig 6.
Ha-ANNEXIN suppresses flg22-triggered upregulation of PTI marker genes in Nicotiana benthamiana.
The upregulation of three PTI marker genes—NbPti5, NbAcre31 and NbGras2—after flg22 treatment in N. benthamiana leaf tissues expressing Ha-ANNEXIN or the negative control eGFP was compared using qPCR, respectively. Each column represents the mean with standard deviation. The column with asterisks indicate a highly statistically significant difference compared with the eGFP negative control by t-test (P<0.01).
Fig 7.
Ha-ANNEXIN can suppress MAPK-triggered cell death.
Ha-ANNEXIN can suppress MKK1 (A)—or NPK1Nt (B)-triggered cell death. Results of the verification of expression of annexin and eGFP by western blotting are shown below. The Necrosis Index of Ha-ANNEXIN and control eGFP followed by MKK1 (A) or NPK1Nt (B) was scored. Each column represents the mean with standard deviation. The column with asterisks indicate a highly statistically significant reduction of the Necrosis Index of Ha-ANNEXIN compared with that of eGFP by t-test (P<0.01).