Reactions of H471 and HHZ in response to PXO99A
Rice introgression line (IL) H471 and its recurrent parent HHZ were evaluated following an inoculation with Xoo PXO99A at the tillering stage. Distinct brown edges along the clipped sites of H471 were visible at 3 days post-inoculation (dpi), and the brown necrosis at the infection site became more apparent at 5 dpi. These observations indicated the IL exhibited a typical HR to Xoo. In contrast, on the infected HHZ leaves, chlorosis was visible at 3 dpi, and water-soaked lesions rapidly spread along the clipped sites at 5 dpi (Fig. 1a). The lesion lengths of H471 and HHZ at 14 dpi were 0.3 ± 0.2 cm and 16.4 ± 2.2 cm, respectively (Fig. 1b). Moreover, PXO99A growth in H471 was markedly lower than that in HHZ as follows: more than 10-fold lower at 2 dpi, more than 20-fold lower at 3 dpi, about 190-fold lower at 4 dpi, and more than 1,600-fold lower at 5 dpi (Fig. 1c). We also examined the ultrastructural changes in H471 and HHZ leaf cells 3 days after an inoculation with PXO99A via transmission electron microscopy (TEM). Before the inoculation, there were no significant structural differences between the mesophyll cells of H471 and HHZ. However, at 3 dpi, the HHZ cell membranes were slightly damaged, and the outlines of the chloroplasts and mitochondria were unclear. In contrast, clear outlines of the whole cell and organelles were visible for H471, and many starch grains were detected (Fig. 1d). These results indicated that H471 is highly resistant to PXO99A (incompatible interaction between H471 and PXO99A), whereas HHZ is highly susceptible (compatible interaction between HHZ and PXO99A).
Protein identification and quantification
The molecular weight range was greater for the identified rice proteins (4,000–469,100 Da) than for the identified Xoo proteins (5,300–195,500 Da) (Additional files 1 and 2). The isoelectric points were 3.5–12.5 and 4.2–12.4 for most rice proteins and Xoo proteins, respectively (Additional files 1 and 2). To improve analytical precision, all quantitative data for peptides identified in multiple fractions were used to quantify proteins. A total of 1,289,153 MS/MS spectra were obtained and were approximately matched to 196,745 known peptide sequences (Additional file 3). Overall, we identified and quantified 46,076 peptides associated with 8,120 different proteins (7,784 rice proteins and 336 Xoo proteins) in the mixed proteomics samples (H471 + PXO99A and HHZ + PXO99A) at 2 and 3 dpi (Additional files 1-4). A clustering analysis involving all identified proteins revealed that samples from the same genetic background clustered in the same groups (Additional file 5).
Differentially abundant proteins between incompatible and compatible interactions
Only unique peptides were considered for quantifying proteins, resulting in the identification of 374 rice DAPs and 117 Xoo DAPs. Additionally, 264 rice DAPs and 3 Xoo DAPs accumulated more in H471 + PXO99A than in HHZ + PXO99A, with a threshold fold-change > 1.5 and p < 0.05 in t-tests, whereas 171 rice DAPs and 116 Xoo DAPs accumulated less in H471 + PXO99A than in HHZ + PXO99A, with a threshold fold-change < 0.67 and p < 0.05 in t-tests (Fig. 2a, 2b, Additional files 6 and 7). Furthermore, 249 rice DAPs and 36 Xoo DAPs were detected between H471 + PXO99A and HHZ + PXO99A at 2 dpi, whereas 262 rice DAPs and 116 Xoo DAPs were detected between H471 + PXO99A and HHZ + PXO99A at 3 dpi. A total of 137 rice DAPs and 35 Xoo DAPs were common between the two time-points (Fig. 2a and 2b). The hierarchical clustering of all rice DAPs indicated that rice DAPs in HHZ at 2 and 3 dpi were clustered in one subgroup, whereas those in H471 at 2 and 3 dpi were in another subgroup (Fig. 2c). The clustering of all Xoo DAPs suggested that Xoo DAPs in H471 at 2 and 3 dpi were clustered in one subgroup, whereas DAPs in HHZ at 2 dpi were grouped with those in H471 (Fig. 2d, Additional file 6). On the basis of protein abundances, the 374 rice DAPs (Additional file 7) were classified into five groups (G-I to V). The proteins in G-III and G-V were more abundant in H471 than in HHZ, whereas the opposite trend was observed for the DAPs in G-II (Fig. 2c). We also extracted the total proteins of HHZ and H471 at 0, 0.5, 1, 1.5, 2, 2.5, 3.5, 4, and 4.5 dpi, and used specific antibodies against four of the obtained DAPs to validate their abundances at different time-points. The western blot results were highly consistent with the proteomic experiments (Fig. 3). However, the data obtained in the present proteomic study was poorly correlated with the results of our previous transcriptomic analyses, implying there are specific mechanisms for maintaining proper levels of transcripts and proteins .
The application of the KOBAS platform to identify enriched GO terms and pathways revealed that Xoo DAPs in H471 were significantly enriched with 14 GO terms, and ribosomal proteins (22.5%) formed the largest set of modulated proteins (Additional file 8). The functional classification of these proteins indicated that most were involved in structural molecule activity and RNA binding. Most of the proteins assigned to the biological process category were involved in translation, protein folding, and generation of precursor metabolites and energy (Additional file 8). The significantly enriched KEGG pathways in Xoo were those related to the ribosome (xop03010, p = 1.4E−14), RNA degradation (xop03018, p = 1.3E−3), methane metabolism (xop00680, p = 9.51E−03), gluconeogenesis (xop00010, p = 1.71E−02), carbon metabolism (xop01200, p = 2.75E−02), and the TCA cycle (xop00020, p = 2.89E−02) (Additional file 9). Regarding the rice DAPs, the three enriched GO terms were secondary metabolic processes, endopeptidase inhibitor activity, and peptidase inhibitor activity, and the five enriched KEGG pathways were biosynthesis of secondary metabolites (osa01110, p = 6.74E−03), phenylalanine metabolism (osa00360, p = 1.23E−02), flavonoid biosynthesis (osa00941, p = 3.52E−02), phenylpropanoid biosynthesis (osa00940, p = 4.31E−02), and vitamin B6 metabolism (osa00750, p = 4.55E−02) (Additional files 8 and 9).
Differentially abundant Xoo proteins between the incompatible and compatible interactions
On the basis of protein functions, the detected Xoo DAPs were grouped into the following categories: type III secretion system (T3SS), transcription activator-like (TAL) effector, nutrient uptake, and uncharacterized protein. In this study, the abundances of six outer membrane proteins (Omps) and nine TonB-dependent receptor-related proteins were significantly different between H471 and HHZ mainly at 3 dpi (12/15) (Fig. 4a). Similarly, one T3SS-related protein (HrpE, PXO_03411), one TAL effector (talC3b, PXO_00505), and a VirK protein (PXO_03361) accumulated much more in HHZ-3d than in HHZ-2d and H471-3d. In this study, the VirK protein (PXO_03361) accumulated less in H471 than in HHZ at 2 and 3 dpi. Additionally, elongation factors (PXO_04524 and PXO_01131) were also less abundant in H471 than in HHZ at 3 dpi (Fig. 4b). Increases in the contents of these Xoo DAPs were more pronounced in HHZ at 3 dpi than at 2 dpi, and the fold-changes of these protein abundances were significantly greater in HHZ-3d vs HHZ-2d than in H471-3d vs H471-2d.
Differentially abundant rice proteins between the incompatible and compatible interactions
In addition to the unknown proteins, the rice DAPs identified in the comparison between H471 and HHZ were grouped into the following five categories: signal transduction, transcription, phytohormone, phytoalexin, and defense response. Hierarchical clustering provided an overview of the differential abundance patterns of the protein types (Fig. 4c-g).
Protein kinases and phosphatases are key co-regulators of protein phosphorylation, and are particularly prominent in signal transduction pathways. In this study, 21 differentially abundant protein kinases and 12 differentially abundant protein phosphatases were identified between the incompatible and compatible interactions (Fig. 4c). Calcium-dependent protein kinases (CDPKs) function as Ca2+ sensors and effectors that relay specific Ca2+ signatures to downstream components via CDPK-dependent protein phosphorylation. In this study, the accumulation of OsCDPK7/OsCDPK13 (LOC_Os04g49510) was more than 200 times greater in H471 than in HHZ at 2 dpi (Fig. 4c, Additional file 7). Likewise, a western blot assay indicated that OsCDPK13 accumulated more in H471 than in HHZ at 2.5 dpi (Fig. 3). The abundances of two CDPK-related kinases (CRKs), LOC_Os04g25060/LOC_Os04g25650 and OsCRK5 (LOC_Os04g56430), were also significantly different between H471 and HHZ. Additionally, the accumulation of a somatic embryogenesis receptor kinase (SERK), OsSERK2 (LOC_Os04g38480), was approximately 10-fold greater in H471 than in HHZ at 2 dpi (Fig. 4c, Additional file 7). The mitogen-activated protein kinase (MAPK) cascades comprise three protein kinase components, MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). The OsMKK4 (LOC_Os02g54600) abundance was lower in H471 than in HHZ at 2 dpi (Fig. 4c, Additional file 7). As indicated in Fig. 3, OsMKK4 accumulated less in H471 than in HHZ. Moreover, it was maintained at a relatively high abundance in HHZ, whereas in H471, it accumulated at 0.5 dpi, peaked at 2 dpi, and then decreased at 4 dpi. In the proteomic assays, there was no obvious difference in OsMPK6 accumulation between HHZ and H471 at 2 and 3 dpi. However, the western blot assay involving the OsMPK6-specific antibody suggested that the accumulation of OsMPK6 significantly increased after HHZ and H471 were inoculated with PXO99A, and this protein accumulated much more in H471 than in HHZ from 0.5 to 4.0 dpi (Fig. 3). Additionally, 14 protein phosphatases differentially accumulated in H471 and HHZ, including PP2C30 (LOC_Os03g16170) and two Ser/Thr protein phosphatases (LOC_Os03g13540 and LOC_Os12g44020).
Transcription factors (TF) play key roles in the large-scale transcriptional reprogramming of plants in response to pathogens. A comparison with HHZ revealed 14 DAPs encoding TFs in H471 infected by Xoo (Fig. 4d), including bZIP, MYB, zinc finger, AP2, homeobox, and HSF family members. Seven and five TFs were more and less abundant, respectively, in H471 than in HHZ at 2 or 3 dpi. The MYB family protein LOC_Os01g74020 accumulated significantly less in H471 than in HHZ at 2 dpi, but more in H471 than in HHZ at 3 dpi. A zinc finger family protein (LOC_Os12g18120) accumulated more and less in H471 than in HHZ at 2 and 3 dpi, respectively (Fig. 4d, Additional file 7). Additionally, two bZIP TFs, OsbZIP23 (LOC_Os02g52780) and LOC_Os03g13614, accumulated much more in H471 than in HHZ at 2 and 3 dpi, respectively (Fig. 4d, Additional file 7). The western blot assay result further confirmed that OsbZIP23 was more abundant in H471 than in HHZ at 2, 2.5, and 3 dpi (Fig. 3). Heat stress TFs (Hsfs) also regulate gene expression in response to environmental stress. In this study, OsHsfA2e (LOC_Os03g58160) and OsVOZ2 (LOC_Os05g43950) accumulated less in H471 than in HHZ at 2 dpi (Fig. 4d, Additional file 7).
Phytohormones are signaling molecules that circulate throughout plants and stimulate responses to various environmental stresses. The abundances of two gibberellin receptors (LOC_Os07g06860 and LOC_Os03g57640) differed between H471 and HHZ. Specifically, LOC_Os07g06860 was less abundant in H471 than in HHZ at both 2 and 3 dpi, whereas LOC_Os03g57640 was more abundant in H471 than in HHZ at 3 dpi. Cytokinin-O-glucosyltransferase 3 (LOC_Os10g09990) was less abundant in H471 than in HHZ at both time-points (Fig. 4e). Notably, the following three PALs accumulated much more in H471 than in HHZ: LOC_Os02g41630 and LOC_Os04g43760 at 2 and 3 dpi, and LOC_Os02g41650 at 2 dpi (Fig. 4e).
Phytoalexin accumulation at an infection site is frequently associated with relatively broad-spectrum antimicrobial activity. Additionally, PAL is a key enzyme in the phenylpropanoid pathway contributing to phytoalexin synthesis, and it is also important for SA synthesis. We observed that several proteins involved in the phenylpropanoid pathway, including Os4CL3 (LOC_Os02g08100), two O-methyltransferases (OMTs) (LOC_Os10g02880 and LOC_Os08g06100), and two caffeoyl-CoA O-methyltransferases (COMTs) (LOC_Os06g06980 and LOC_Os08g38900), were considerably more abundant in H471 than in HHZ at one or both time-points after inoculation (Fig. 4f). Moreover, two DAPs associated with the shikimate pathway were identified. The accumulation of chorismate mutase (LOC_Os01g55870) was lower in H471 than in HHZ at 2 dpi, but higher at 3 dpi. In contrast, shikimate kinase (LOC_Os02g51410) accumulated much more in H471 than in HHZ at 2 dpi, but less at 3 dpi. Chalcone-flavonone isomerase (LOC_Os11g02440), which is related to sakuranetin biosynthesis, also accumulated more in H471 than in HHZ at 3 dpi (Fig. 4f). Furthermore, three proteins (LOC_Os03g15050, LOC_Os01g07730, and LOC_Os03g27230) involved in phosphoenol pyruvate biosynthesis accumulated differently in H471 and HHZ. The abundance of LOC_Os03g15050 was lower in H471 than in HHZ at 3 dpi, whereas the abundances of LOC_Os01g07730 and LOC_Os03g27230 were higher in H471 than in HHZ at 2 and 3 dpi. (Fig. 4f).
Three and two defense-related proteins were identified as less and more abundant, respectively, in H471 than in HHZ plants inoculated with Xoo. Two NB-ARC domain-containing protein family members (LOC_Os11g44990 and LOC_Os11g45090) and an NBS-LRR disease resistance protein (LOC_Os11g38580) accumulated less in H471 than in HHZ. Both OsPR1b (LOC_Os01g28450) and a harpin-induced protein (LOC_Os12g06220) accumulated much more in H471 than in HHZ. Notably, the accumulation of LOC_Os12g06220 was more than 160-times higher in H471 than in HHZ at 2 dpi (Fig. 4g, Additional file 7). Additionally, a harpin-induced protein (LOC_Os04g33990) accumulated more and less at 2 and 3 dpi, respectively, in H471 than in HHZ (Fig. 4g).
Abundances of proteins associated with SA signaling during interactions between rice and Xoo
To further investigate whether SA contributes to the disease resistance of H471, we analyzed the abundances of the SA-inducible OsPR1a and OsPR1b. Western blot assays indicated that in plants inoculated with PXO99A, OsPR1a levels increased at 1 and 0.5 dpi in HHZ and H471, respectively, and were much higher in H471 than in HHZ from 2.5 to 4 dpi (Additional file 10a). Because the accumulation of OsPR1b was too low to detect, we were unable to quantify its abundance in HHZ, and only unclear bands were detected at 2.5 and 4.5 dpi for H471 (Additional file 10a). After the inoculation with PXO99A, the SA content in H471 increased rapidly and was double that in HHZ at 1.5 dpi. The SA content was much higher in H471 than in HHZ at 2.5 and 3.5 dpi, and it decreased in HHZ at 4.5 dpi (Fig. 5). These results suggested that the SA signaling pathway was activated in response to the Xoo infection in H471.