Respiratory Burst Oxidase Homologs RBOHD and RBOHF as Key Modulating Components of Response in Turnip Mosaic Virus—Arabidopsis thaliana (L.) Heyhn System

Abstract

Turnip mosaic virus (TuMV) is one of the most important plant viruses worldwide. It has a very wide host range infecting at least 318 species in over 43 families, such as Brassicaceae, Fabaceae, Asteraceae, or Chenopodiaceae from dicotyledons. Plant NADPH oxidases, the respiratory burst oxidase homologues (RBOHs), are a major source of reactive oxygen species (ROS) during plant–microbe interactions. The functions of RBOHs in different plant–pathogen interactions have been analyzed using knockout mutants, but little focus has been given to plant–virus responses. Therefore, in this work we tested the response after mechanical inoculation with TuMV in ArabidopsisrbohD and rbohF transposon knockout mutants and analyzed ultrastructural changes after TuMV inoculation. The development of the TuMV infection cycle was promoted in rbohD plants, suggesting that RbohD plays a role in the Arabidopsis resistance response to TuMV. rbohF and rbohD/F mutants display less TuMV accumulation and a lack of virus cytoplasmic inclusions were observed; these observations suggest that RbohF promotes viral replication and increases susceptibility to TuMV. rbohD/F displayed a reduction in H2O2 but enhanced resistance similarly to rbohF. This dominant effect of the rbohF mutation could indicate that RbohF acts as a susceptibility factor. Induction of hydrogen peroxide by TuMV was partially compromised in rbohD mutants whereas it was almost completely abolished in rbohD/F, indicating that these oxidases are responsible for most of the ROS produced in this interaction. The pattern of in situ H2O2 deposition after infection of the more resistant rbohF and rbohD/F genotypes suggests a putative role of these species on systemic signal transport. The ultrastructural localization and quantification of pathogenesis-related protein 1 (PR1) indicate that ROS produced by these oxidases also influence PR1 distribution in the TuMV-A.thaliana pathosystem. Our results revealed the highest activation of PR1 in rbohD and Col-0. Thus, our findings indicate a correlation between PR1 accumulation and susceptibility to TuMV. The specific localization of PR1 in the most resistant genotypes after TuMV inoculation may indicate a connection of PR1 induction with susceptibility, which may be characteristic for this pathosystem. Our results clearly indicate the importance of NADPH oxidases RbohD and RbohF in the regulation of the TuMV infection cycle in Arabidopsis. These findings may help provide a better understanding of the mechanisms modulating A.thaliana–TuMV interactions.

FERONIA receptor kinase-regulated reactive oxygen species mediate self-incompatibility in Brassica rapa

“…Next, we speculated that the rapid induction of high ROS levels after self-pollination is utilized to arrest self-pollen, similar to the ROS burst in a wide range of plant-pathogen interactions to inhibit the invading pathogens. [49][50][51][52][53][54] To prove this, we carried out a ''stigma transfer experiment'' (Figure S3D) by first pollinating stigmas with self-pollen for 0-60 min to induce different levels of ROS elevation and then transferring them onto 3 mM CuCl 2 medium for further cultivation. Compared to the typical SI phenotypes in stigmas that were not transferred, an average of 80 self-pollen tubes were salvaged and grew into the SI 0 stigmas, which were immediately transferred after self-pollination, but only 42 and 10 self-pollen tubes grew into the SI 15 and SI 30 stigmas (Figures 3B and S3F).…”

Modulation of Expression of PVYNTN RNA-Dependent RNA Polymerase (NIb) and Heat Shock Cognate Host Protein HSC70 in Susceptible and Hypersensitive Potato Cultivars

“…For a more precise estimation, the corrected mean OD 405nm values were computed as described in [35] and used to compare the relative levels of virus presence/concentration in plants. The cut-off point was calculated using a formula suggested by Bioreba (Switzerland) [37] and Otulak-Kozieł et al [38].…”

“…The calculated cut-off point value was 0.135. The readings at OD 405nm were compared to the calculated cut-off point, and all OD 405nm values greater than 0.135 were considered to be positive (i.e., confirmed the presence of the virus) [38]. The significant threshold/cut-off point values obtained after performing DAS-ELISA confirmed the presence of the virus in all the inoculated potato plants.…”

“…The expression of PVY NTN -NIb and HSC70-8 genes was investigated in both types of potato cultivars. The S. tuberosum StEf1α gene (GeneID: AB061263) was used as the reference gene, as suggested in [38]. The primers were designed using Primer3 software (version 0.4.0; Primer3Plus, Free Software Foundation, Inc., Boston, MA, USA).…”

“…The leaf samples (mock-and virus-inoculated) of potato plants at 3, 7, and 21 dpi were fixed, embedded in epoxy resin (EPON, Fluka, Switzerland), and polymerized at 60 • C for 24 h as previously described [38]. Then the leaf sections were mounted on Formvar-coated nickel grids and treated according to the procedure of immunogold localization [33,34].…”