P3N-PIPO (P3 N-terminal fused with Pretty Interesting Potyviridae ORF), the movement protein of potyviruses, is expressed as a translational fusion with the N-terminus of P3 in potyviruses. As reported in previous studies, P3N-PIPO is expressed via transcriptional slippage at a conserved G₂A₆ slippery site in the genus Potyvirus. However, it is still unknown whether a similar expression mechanism of P3N-PIPO is used in the other genera of the family Potyviridae. Moreover, due to the extremely low expression level of P3N-PIPO in natural virus-infected plants, the peptides spanning the slippery site which provide direct evidence of the slippage at the protein level, have not been identified yet. In this study, a potato virus X (PVX)-based expression vector was utilized to investigate the expression mechanism of P3N-PIPO. A high expression level of the P3N-PIPO(WT) of turnip mosaic virus (TuMV, genus Potyvirus) was observed based on the PVX expression vector. For the first time, we successfully identified the peptides of P3N-PIPO spanning the slippery site by mass spectrometry. Likewise, the P3N-PIPO(WT) of wheat yellow mosaic virus (WYMV, genus Bymovirus) was also successfully expressed using the PVX expression vector. Integrated proteome and transcriptome analyses revealed that WYMV P3N-PIPO was expressed at the conserved G₂A₆ site through transcriptional slippage. Moreover, as revealed by mutagenesis analysis, Hexa-adenosine of the G₂A₆ site was important for the frameshift expression of P3N-PIPO in WYMV. According to our results, the PVX-based expression vector might be used as an excellent tool to study the expression mechanism of P3N-PIPO in Potyviridae. To the best of our knowledge, this is the first experimental evidence dissecting the expression mechanism of a bymovirus P3N-PIPO in the experimental host Nicotiana benthamiana.

Similarly to other potyvirids, the bymovirus wheat yellow mosaic virus (WYMV) encodes a P3N-PIPO protein that is expressed by frameshifting occurring within the open reading frame of the P3 protein. P3N-PIPO is known to be essential for the cell-to-cell movement of several potyviruses, but this has not yet been confirmed for the WYMV. Here, we show that the WYMV P3N-PIPO protein influences disease symptom formation. Infection of Nicotiana benthamiana plants with a potato virus X (PVX)-based vector carrying the WYMV P3N-PIPO gene induced more severe disease symptoms and resulted in higher virus accumulation levels than did infection with PVX lacking the P3N-PIPO gene. N. benthamiana P3N-PIPO-interacting proteins were identified through co-immunoprecipitation (Co-IP) coupled with LC-MS/MS (mass spectrometry), and the interaction between P3N-PIPO and the N. benthamiana receptor-like kinase NbRLK6 was further verified by Co-IP and bimolecular fluorescence complementation (BiFC) of transiently-expressed proteins. Furthermore, our investigation showed that the disease symptom severity and accumulation level of PVX-P3N-PIPO were decreased in N. benthamiana plants when NbRLK6 expression was reduced by tobacco rattle virus-induced gene silencing.

Oryza meyeriana is a wild species of rice with high resistance to Xanthomonas oryzae pv. oryzae (Xoo), but the detailed resistance mechanism is unclear. Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA) is an important enzyme that regulates photosynthesis by activating Rubisco. We have previously reported that Xoo infection induced the relocation of RCA from the chloroplast stroma to the thylakoid membrane in O. meyeriana, but the underlying regulating mechanism and physiological significance of this association remains unknown. In this study, “H₂O₂ burst” with rapid and large increase in the amount of H₂O₂ was found to be induced by Xoo invasion in the leaves of O. meyeriana. 3, 3-diaminobenzidine (DAB) and oxidative 2, 7-Dichlorodi-hydrofluorescein diacetate (H₂DCFDA) staining experiments both showed that H₂O₂ was generated in the chloroplast of O. meyeriana, and that this H₂O₂ generation as well as Xoo resistance of the wild rice were dramatically dependent on light. H₂O₂, methyl viologen with light, and the xanthine-xanthine oxidase system all induced RCA to associate with the thylakoid membrane in vitro, which showed that H₂O₂ could induce the relocation of RCA. In vitro experiments also showed that H₂O₂ induced changes in both the RCA and thylakoid membrane that were required for them to associate and that this association only occurred in O. meyeriana and not in the susceptible cultivated rice. These results suggest that the association of RCA with the thylakoid membrane helps to protect the thylakoid membrane against oxidative damage from H₂O₂. Therefore, in addition to its universal function of activating Rubisco, RCA appears to play a novel role in the resistance of O. meyeriana to Xoo.

Xanthomonas oryzae pv. oryzae (Xoo) is the causative agent of rice bacterial blight (RBB), resulting in substantial harvest losses and posing a challenge to maintaining a stable global supply. In this study, Xoo strains isolated from Shaoxing, Quzhou, and Taizhou, where RBB occurred most frequently in Zhejiang Province in 2019, were selected as the subjects of research. Three isolated pathogenic bacteria of ZXooS (from Shaoxing), ZXooQ (from Quzhou), and ZXooT (from Taizhou) were all identified as novel Xoo strains. These novel strains demonstrate greater virulence compared to Zhe173, the previous epidemic Xoo strain from Zhejiang Province. Subsequent genomic sequencing and analysis revealed that there existed significant differences in the genome sequence, especially in effector genes corresponding to some known rice resistance (R) genes between the novel strains and Zhe173. The sequence alignment of avirulent genes (effector genes) indicated that nucleic and amino acid sequences of AvrXa5, AvrXa7, AvrXa10, and AvrXa23 in the novel strains varied prominently from those in Zhe173. Interestingly, it seemed that only the genome of ZXooQ might contain the AvrXa3 gene. In addition, the phylogenetic analysis of 61 Xoo strains revealed that the novel strains were situated in a distinct evolutionary clade separate from Zhe173. These results here suggest that the emergence of novel Xoo strains may lead to resistance loss of some R genes used in commercial rice varieties, potentially serving as one of the factors leading to RBB resurgence in Zhejiang Province, China.