Discussion
In this study, we carried out a comprehensive transcriptome study of
susceptible and resistant rice cultivars over a 48 h time course of
infection with the blast fungus M. oryzae . Our data showed that
high-amplitude transcriptional reprogramming was kicked off very quickly
in rice-M. oryzae compatible and incompatible interaction. Large
numbers of DEGs were identified, and a core set of genes involved in
rice stress responses were defined. Furthermore, our study not only
provided an overview of transcriptional reprogramming during
rice-M. oryzae interactions but also identified and functionally
validated three novel players in rice blast resistance.
A number of rice blast resistance genes and signaling regulators have
been mapped and identified after decades of study (Li et al., 2019; Liu
et al., 2014). However, the full regulatory network and immune signaling
components remain elusive. Here we designed a comprehensive RNA-seq
experiment to study the transcriptional profiles of rice-M.
oryzae compatible (susceptible Nipponbare) and incompatible (resistant
Hui1586) interactions. Four time points were included in the experiment,
from very early at 12 hpi to later at 48 hpi with 12 h intervals between
each point. Because the circadian clock controls the expression of
~ 30% of the transcriptome in plants (Harmer, 2009),
mock-treatment (H2O) samples were included as controls
at each time point to ensure a proper comparison for identifying DEGs.
Our RNA-seq data showed that the circadian clock is the major factor
determining the transcription dynamics (Fig. 1A, H2O
treatment). Thus, it was essential to compare M. oryzae treatment
data with the mock data to identify DEGs during the rice response to
blast fungi. In this way, thousands of DEGs were identified at each time
point, far exceeding the numbers in previous reports (Wei et al., 2013;
Y. Zhang et al., 2016). In total, the expression of
~25% of the rice transcriptome was affected by M.
oryzae treatment (Fig. 2C and 2D), indicating that great
transcriptional changes occurred in rice challenged with blast fungus.
Remarkably, the largest number of DEGs (6808 upregulated and 2895
downregulated, 9703 in total) was observed in the resistant Hui1586 at
12 hpi of blast fungus. Compared with the smaller number of DEGs in the
susceptible Nipponbare (4680 upregulated and 2045 downregulated genes),
we concluded that the resistant cultivar induced active defense more
quickly and strongly than the susceptible cultivar. Unexpectedly, the
number of DEGs declined in Hui1586 to 2849 (2224 upregulated and 625
downregulated) at 24 hpi, which was ~1/3 of the DEG
number at 12 hpi. This result suggested that the strongest
transcriptional reprogramming occurred before 24 hpi, and the critical
time period for M. oryzae invasion was probably less than 12
hours. RNA-seq data for more time points, earlier than 12 hpi, is needed
to determine the critical time points for rice-M. oryzaeinteractions.
In addition to transcriptional reprogramming, global translational
reprogramming has been shown to be another fundamental regulatory layer
of plant immunity (Xu et al., 2017). A consensus sequence, R-motif,
present on a large number of messenger RNA, regulates translation in
response to PTI induction (Xu et al., 2017). A latest study has shown
that plants protect stem cells against viral infection through impairing
global protein synthesis and limiting the replication and spread of the
virus (Wu et al., 2020). In our transcriptome analysis, we found that
among the common upregulated DEGs at 12 hpi in both Nipponbare and
Hui1586, “ribosome” was the most significantly enriched pathway in the
KEGG analysis, and “translation” was the most significantly enriched
term in the GO analysis (Fig. 4C). These results strongly indicated that
the protein translation machinery was regulated during the rice response
to M. oryzae . Consistent with our results, a previous
transcriptome study of rice Pi21- silenced plants infected byM. oryzae showed that “ribosome” is the third most enriched
pathway compared with Nipponbare plants (Y. Zhang et al., 2016).Pi21 encodes a cytoplasmic proline-rich protein that negatively
regulates rice blast resistance, and silencing of this gene results in
enhanced resistance to blast fungi (Fukuoka et al., 2009). However,
enrichment of the “ribosome” pathway was only observed at 12 hpi, not
24, 36, or 48 hpi, indicating that the regulation of protein synthesis
in rice responses to M. oryzae was transient. Protein synthesis
is a high-energy consumption process in living cells. It consumes
approximately two-thirds of the total energy produced by a rapidly
growing Escherichia coli cell (Jewett, Miller, Chen, & Swartz,
2009). Plant defense also imposes a substantial demand of resources and
energy, which negatively impact growth (Huot et al., 2014). Activation
of defense would decrease the overall pool of energy reserves through
diminishing photosynthesis. It is possible that the demand of energy
during the quick activation of plant immunity might reduce energy for
the ribosome, the protein translation factory, slowing down the
translation globally. As a negative feedback, the transcription of
ribosome-related genes was upregulated accordingly. Further biochemical
experiments are required to examine whether the global protein
translation was transiently suppressed during the activation of rice
immunity. However, in agreement with this hypothesis, protein synthesis
inhibitor cycloheximide treatment of Arabidopsis induces
transcriptional reprogramming similar to pathogen treatment (Navarro et
al., 2004).
Among more than 10 thousand DEGs identified from four time points, only
1464 and 578 DEGs were shared by all four time points in Nipponbare and
Hui1586, respectively, indicating that the transcriptome in the rice
response to blast fungus infection was changing dynamically. A small set
of DEGs shared by Nipponbare and Hui1586 at all time points were
identified. These common DEGs might be important for driving overall
immune transcriptional reprogramming. Indeed, only defense-related
signaling and metabolism pathways were enriched in the common DEG group,
and therefore, these genes were considered the “core” genes inM. oryzae- induced transcriptional reprogramming. Expressions of
most of the core genes were also induced by other pathogens, such as the
fungal U. virens and bacterial X. oryzae (Fig. 6A).
Furthermore, the core genes were largely repressed during drought stress
(Fig. 6B). Thus, the core genes were likely common genes involved in
rice defense against pathogens and abiotic stress responses. Supporting
this conclusion, among 14 TFs from the core genes, 5 TFs have been
functionally characterized as important regulators of disease
resistance, and 2 TFs have been reported to be involved in rice
tolerance to temperature stress. We speculate that the core gene set
contains convergence points between biotic and abiotic stress signaling
pathways.
In this study, we identified a peroxidase gene Perox4 that plays
a negative role in rice resistance to blast fungus (Fig. 8). The main
role of peroxidase in plant immune responses is to aid in maintaining
hydrogen peroxide (H2O2), the most
stable ROS, at the appropriate level, which is toxic to plant cells at
high concentrations (Qi, Wang, Gong, & Zhou, 2017). Three rice
peroxidases (Os05g0135200 , Os10g0536700 , andPerox3 ) have been reported to contribute to BSR-D1, a
C2H2-type transcription factor-mediated
susceptibility to blast disease (Li et al., 2017; Zhu et al., 2020).
BSR-D1 induces the expression of these peroxidase genes by direct DNA
binding. An allele, Bsr-d1, in the rice cultivar Digu confers
broad-spectrum resistance to M. oryzae (Li et al., 2017; Zhu et
al., 2020). Notably, the expression of Perox4 is
compromised in bsr-d1 knockout plants (Zhu et al., 2020),
suggesting it is one of the target of BSR-D1. However, it remains
inconclusive whether BSR-D1 can bind to the Perox4 promoter (Zhu
et al., 2020). In our RNA-seq data, Perox3 was upregulated in
Nipponbare and Hui1586, while the mRNA of Os05g0135200 andOs10g0536700 were barely detectable even with M.
oryzae -treatment in both cultivars. In addition, another 4 peroxidase
genes were upregulated by M. oryzae in Nipponbare and Hui1586
(Supplemental table 7). The peroxidase genes were proposed to be
hijacked by M. oryzae through activation of the BSR-D1gene to counter the ROS burst induced by M. oryzae infection. It
is reasonable to speculate that as an important susceptibility factor,Perox4 might be hijacked by blast fungi to suppress host immune
responses.
In addition to ROS-scavenging enzymes, low-molecular mass antioxidants,
including glutathione, ascorbate, carotenoids and (MTs), are involved in
ROS maintenance (Qi et al., 2017; Yang, Wu, Li, Ling, & Chu, 2009). MTs
are small, cysteine-rich, metal-binding proteins that are involved in
metal homeostasis and detoxification in both plants and animals (Yang et
al., 2009; Zimeri, Dhankher, McCaig, & Meagher, 2005). It has been
shown that OsMT1a improves drought tolerance when overexpressed
in rice by not only participating in ROS scavenging but also regulating
the expression of zinc finger-type TFs via the regulation of
Zn2+ homeostasis (Yang et al., 2009). However, unlike
the ROS-scavenging enzyme Perox4 , simultaneous knockout ofOsMT1a and its close homolog OsMT1b in Nipponbare further
enhanced susceptibility to blast fungus (Fig. 9). Thus, OsMT1positively regulated drought tolerance and blast disease resistance. DNA
motifs of several zinc finger-type TFs were significantly enriched in
the promoter of 321 core genes (Fig. 7C and Supplemental dataset 11),
suggesting that OsMT1 might regulated blast disease resistance in
a manner similar to the regulation of drought stress resistance, partly
through zinc finger-type TFs.
In summary, this study provides a high-quality, comprehensive RNA-seq
data set for rice-M. oryzae interactions and enhances our
understanding of the transcriptional networks in rice immune responses
to the blast fungus, highlighting possible candidate genes that may play
important roles in rice disease resistance and abiotic stress tolerance.