Rice blast fungus up-regulates the expression of Pik-H4
As a resistant gene pair, Pik1-H4 collaborates with Pik2-H4 to participate in rice blast immunity. The presence of W-boxes within the PPik-H4 , which has been documented to confer promoter responsiveness to fungal or bacterial infections(In et al. , 2020), suggests a potential pathogen-induced expression mechanism for Pik-H4 . To identify whether Pik-H4 responds to rice blast fungus, the Pik-H4 NILs leaves were spray inoculated with rice blast fungus carrying AvrPik-E. We found that the Pik-H 4 expression levels peaked at 24 hours post-inoculation (hpi) through qRT-PCR analysis (Figure S4A, C), a pattern distinct from the mock treatment (Figure S4B). Notably, this peak coincided with the onset ofM. oryzae invasion, wherein the penetration peg of the fungus invades rice cells around 24 hpi, followed by the subsequent spread of invasive hyphae between cells(Yan and Talbot, 2016). The temporal alignment of Pik-H4 expression peak with M. oryzaeinvasion highlights its up-regulation at the initiation of ETI.
Since Nip is infectious to AvrPik-E, theRFP ::PPik-H4 ::GFP /Nip plants were also inoculated and observed in planta . Visible lesion areas emerged on 2 days post-inoculation (dpi) in leaves, and our observations continued as these lesions evolved. As the lesions expanded, the leaf mesophyll became increasingly transparent, enabling laser penetration. As a result, fluorescence intensity data from 4 dpi onward was not collected for this study. The PPik-H4 activity was measured from the lesion area to the peripheral area along the dotted line in Figure 4A. Within the lesion area, the promoter activity of Pik1-H4 and Pik2-H4 was lower than the average levels but rose at the edge of the lesion (Figure 4B). The RFP signal was higher than the average levels of leaves from the lesion edge to the lesion peripheral region while the GFP signal dropped to the normal state. These findings suggest that Pik-H4is responsive to rice blast fungus and a putative signaling cascade operates from the lesion area to its periphery. Similar trends were observed at the lesion edge as the lesion expanded in the following days post-inoculation (Data not shown). We observed the promoter activity changes of M. oryzae at the biotrophic and necrotrophic stages within vascular bundles (Figure 4C). During the early stage of hypha growth, Pik-H4 displayed no responsiveness (Figure 4D). However, as the hyphae advanced along the vascular bundles, PPik-H4 activity exhibited up-regulation. Conversely, at the distal end of lesions by 4 dpi, the transcription levels of PPik-H4 demonstrated no significant difference compared to the mock treatment. Interestingly, the fluorescence intensity of spreading hyphae showed no difference between the hyphal region and its surroundings, while at the biotrophic stage, the intensity was significantly higher than the peripheral area (Figure 4E). As for cell scale, the activity of PPik-H4in both mesophyll cells and vascular cells markedly increased upon the appearance of M. oryzae (Figure 4F-I), although the linear correlation between Pik1-H4 andPik2-H4 promoter activity was lost (Figure 4J, K). Notably, no discernible PPik-H4 activity difference was observed in cells harboring M. oryzae spores or hyphae and their adjacent cells (Figure S4G).
Given that the Pik-H4 gene pair demonstrated co-expression in various tissues, rice blast was inoculated in leaf sheaths for PPik-H4 activity analysis. In Pik-H4 NILs,Pik-H4 conferred rice blast resistance in leaf sheaths (Figure S4D). PPik-H4 exhibited responsiveness to the rice blast infection, leading to the up-regulation of Pik-H4 in leaf sheaths at 24 hpi in contrast to the mock treatment (Figure S4E, F). Taken together, the Pik-H4 gene pair was rice blast inducible.