Proteomic strategies have recently been successfully applied to analyze plant disease resistance. A number of proteins associated with BB resistance responses in rice have been identified. However, little information is available on the global changes in pathogen proteins during plant–pathogen interactions. In this study, the DAPs of the plant host and pathogen were examined simultaneously. Specifically, quantitative proteomic technology was used to compare incompatible (H471 and PXO99A) and compatible (HHZ and PXO99A) interactions. The differences in the proteome profiles of rice and Xoo between the incompatible and compatible interactions revealed interesting molecular mechanisms underlying the broad-spectrum resistance of rice to BB.
The abundances of proteins related to Xoo invasion and growth in rice were lower during the incompatible interaction than during the compatible interaction
Entry into host cells is a crucial step in bacterial infections. Bacterial pathogens use diverse virulence-related factors, including the T3SS, for host cell binding, the triggering of membrane ruffles, and cooperative invasions. The Omps of Gram-negative bacteria are essential for bacterial survival during eukaryotic cell invasions [22, 23]. The OmpA protein is required for bacterial infection/colonization and cellular adhesion/development in host cells . Bacterial TonB-dependent receptors, which are involved in the uptake of nutrients from the surrounding environment, and extracellular enzymes are also important for the interactions between Xoo and rice . An elongation factor is essential for viability and is required for protein synthesis. In this study, DAPs related to six Omps, nine TonB-dependent receptors, and two elongation factors were detected, and all of them accumulated less in H471 than in HHZ. These results suggested that PXO99A invasion and growth were significantly more inhibited in the incompatible interaction than in the compatible interaction. The dramatically decreased Xoo population in H471 was consistent with the differential protein abundances.
The T3SS, encoded by hrp genes, plays an important role in the interaction between Xoo and rice because it injects T3 effectors into plant cells . The Xoo T3 effectors include two collections of proteins, namely TAL effectors and Xanthomonas outer proteins (Xop). The TAL effectors can recognize and bind to specific DNA sequences within the promoters of corresponding host genes, resulting in the transcriptional activation of the genes mediating disease susceptibility or resistance . In the current study, HrpE (PXO_03411) accumulated less in H471 than in HHZ. Notably, the T3 effectors talC3b (PXO_00505) and OsVOZ2 (LOC_Os05g43950) also accumulated less in H471 than in HHZ. A previous study proved that interactions between XopNKXO85 and OsVOZ2 increase rice susceptibility to Xoo . However, in the present study, we were unable to identify the target gene of talC3b based on the list of rice DAPs. Additionally, the PXO99A XopN protein abundance was similar in H471 and HHZ at the two examined time-points. This might have been due to a shift in the timing of gene expression as the host-pathogen interaction developed. We speculated that the effectors talC3b and XopN may serve as signaling molecules that modulate the host response to infection when they are released in plant tissues, wherein they activate their target genes whose protein products contribute to the susceptibility of the host plants in a compatible interaction. In contrast, these proteins do not mediate a susceptible response in incompatible interactions because of their lower abundances. Clarifying the functions of the two effectors and their host targets will be a key step for further characterizing the interaction between H471 and Xoo.
Diverse rice proteins related to signal transduction and transcriptional regulation are involved in the plant-pathogen interaction
During plant-pathogen interactions, plants not only perceive and prevent the pathogen from invading, they also initiate defense responses by activating multi-component systems via modulated expression or abundance and/or post-translational modifications of the associated proteins. After the inoculation with Xoo, the accumulation of diverse categories of rice protein kinases and TFs differed significantly between the incompatible and compatible interactions.
Receptor-like kinases (RLKs) reportedly regulate defense processes . On the basis of amino acid sequence and structural differences, RLKs have been categorized into several subfamilies, including leucine-rich repeat RLKs (LRR-RLKs), cysteine-rich repeat RLKs (CRKs), domain of unknown function 26 RLKs, and S-domain RLKs, among others . As common key signaling molecules, CRKs influence diverse stress responses. For example, OsCRK5 expression is up-regulated in rice in response to the blast fungus or brown planthopper [30, 31]. In the current study, OsCRK5 was more abundant in H471 than in HHZ, in contrast to another CRK, LOC_Os04g25060/LOC_Os04g25650, which was less abundant in H471 than in HHZ. Genes encoding CDPKs are critical for abiotic stress responses [32, 33], but they are also expressed in response to biotic stresses in plants . A previous study indicated that OsCDPK7 (OsCDPK13) expression can be induced by an exposure to chilling stress and high salt concentrations . In the present study, OsCDPK7/OsCDPK13 (LOC_Os04g49510) was significantly more abundant in H471 than in HHZ. The MAPK cascade has been implicated in signaling during plant defense responses to pathogens and various environmental stimuli . In rice, chitin elicitors may activate OsMPK1, OsMPK5, OsMKK4, and the OsMKK4-OsMPK6 cascade to regulate defense activities, including antimicrobial compound biosynthesis, and induce plant cell death [37, 38]. In the current study, OsMKK4 and OsMPK6 accumulated much more in H471 than in HHZ at 1.5–3 dpi. Moreover, 14 phosphatase DAPs, including PP2C30 and two Ser/Thr protein phosphatases, accumulated differently in H471 and HHZ. A previous investigation revealed that PP2C30 interacts with the ABA receptor PYL/RCAR5 and SAPK2 to regulate ABA-dependent gene expression . It also functions as an upstream regulator of HOX12 expression, acting directly through EUI1 to control panicle exsertion in rice .
The bZIP TF family participates in plant responses to abiotic stress via the ABA signaling pathway. For example, OsbZIP71 RNAi knockdown transgenic lines are highly sensitive to salt, PEG-induced osmotic stress, and ABA . Additionally, OsbZIP23 is a central regulator of ABA signaling and biosynthesis as well as drought tolerance in rice . Moreover, OsbZIP46 positively regulates ABA signaling and drought tolerance in rice, depending on whether it is activated . Our data indicated that two bZIP TFs, namely OsbZIP23 (LOC_Os02g52780) and LOC_Os03g13614, accumulated much more in H471 than in HHZ at 2 and 3 dpi. Furthermore, western blot assays confirmed that OsbZIP23 accumulated more in H471 than in HHZ. These results imply that bZIP TFs are important for the incompatible interaction between rice and Xoo.
Phytoalexin and SA biosynthetic pathways were more enhanced in rice during the incompatible interaction than during the compatible interaction following the Xoo infection
In response to pathogens, plants may produce many phytoalexins, including the flavonoid sakuranetin . Sakuranetin is the only known phenolic phytoalexin in rice, and phenylamides are involved in its biosynthesis. The accumulating phenylamides or sakuranetin in plants help reinforce the cell wall and are toxic to pathogens at the infection site [6, 44–46]. Additionally, SA is a small phenolic compound that has an important regulatory role in multiple physiological processes. There are two SA biosynthetic pathways in plants: one involves cinnamate and includes a reaction catalyzed by PAL, whereas the other requires chorismate and includes a reaction catalyzed by ICS . The accumulation of SA always coincides with the up-regulated expression of antimicrobial PR genes, which enhances disease resistance responses . Methylated derivatives of SA spread from the infected tissue to distal tissues, thereby inducing systemic acquired resistance .
Phenylalanine ammonia-lyases are key enzymes in the phenylpropanoid pathway, which mediates the biosynthesis of the flavonoid-type phytoalexin, sakuranetin, and SA . In the current study, 14 DAPs associated with phenolic phytoalexin biosynthesis were detected. Three PALs (OsPALs) accumulated much more in H471 than in HHZ, whereas no proteins contributing to the chorismite-based SA biosynthetic pathway were identified as a DAP. Furthermore, the accumulation of OsPR1b was about 2-fold higher in H471 than in HHZ. These results suggest that the phytoalexin and SA biosynthetic pathways were activated faster during the incompatible interaction than during the compatible interaction, implying that phytoalexins and SA are involved in the HR of H471 (Fig. 5).