Environment changes induced by unfavorable growth conditions inevitably affect crop yield every year. Indeed, under long-term natural selection pressures, sessile plants have evolved a complex set of molecular and physiological mechanisms to cope with abiotic/biotic stresses. However, poverty, climate change, population growth, the COVID-19 pandemic, and even other potential risk forces agriculture system to evolve in a high-production and sustainable path. To adequately address the food security challenge, plant breeders expanding numerous tools and mining molecular resources to develop new improved crop varieties (Lenaerts et al., 2019; Zaidi et al., 2019). Moreover, the current emergence of the CRISPR-Cas systems in many crops provides an effective and straightforward suite to precisely and efficiently process genome editing (Zaidi et al., 2019). Precise genome editing requires an in-depth understanding of the underlying mechanisms with desired breeding goals.

Rice (Oryza sativa L.) stands as a cornerstone in global agriculture, serving as a staple food for a significant proportion of the world’s population. However, this crucial crop encounters a multitude of threats from both biotic and abiotic stresses. Among the major concerns is bacterial blight (BB), incited by the gram-negative bacterial pathogen Xanthomonas oryzae pv. Oryza (Xoo), and temperature-related stresses, including heat and cold stresses (Khush, 2005). Deciphering the intricate signaling pathways that rice employs in response to these concurrent stresses, and their intricate crosstalk, is pivotal for developing crop varieties with enhanced tolerance to these combined stresses. Atkinson and Urwin (2012) emphasized the need for more holistic approaches to plant stress research, considering the combined effects of multiple stress factors that crops face in the field, as opposed to studying individual stresses in isolation (Atkinson and Urwin, 2012). In Arabidopsis, the involvement of the NPR1 (Nonexpresser of Pathogenesis-Related Genes 1) gene in combined stress responses has been highlighted. NPR1, a key regulator in salicylic acid-mediated resistance to pathogens, also confers tolerance to drought stress, signifying its role in the crosstalk between biotic and abiotic stress signaling pathways (Zhang et al., 2019). However, the majority of existing research has been centered on individual stress responses, leaving the cumulative impact of combined stressors somewhat understudied.

bHLH (basic Helix-Loop-Helix) transcription factors constitute the second largest families after the MYBs in plants and are involved in a broad range of biological processes. This group of proteins modulates both disease resistance and temperature stress tolerance. The bHLH transcription factors PIF4 and PIF5, for example, are known to mediate plant responses to high temperatures by influencing auxin signaling pathways (Leivar and Quail, 2011). Conversely, the bHLH transcription factor ICE1 is documented to govern the CBF pathway, subsequently enhancing cold tolerance in plants (Chinnusamy et al., 2007). The functionality of bHLH transcription factors under the influence of combined temperature stresses, and potentially in conjunction with biotic stress such as bacterial blight, is an area that necessitates further exploration.

Indeed, extensive research of basic helix-loop-helix (bHLH) transcription factors in Oryza sativa has delineated the function of key members such as OsIRO2, OsbHLH006, and OsbHLH062, each of which has integral roles in iron homeostasis, abiotic stress responses, and disease resistance, respectively (Ogo, 2006; Seo et al., 2011; Liu et al., 2015). However, given the considerable number and diversity of bHLH transcription factors in rice, it is evident that a vast array of these proteins remains functionally uncharacterized (Toledo-Ortiz et al., 2003; Pires and Dolan, 2010; Guo et al., 2021; Wang et al., 2023). The intricacy and multifariousness of bHLH proteins substantiate the likelihood of unveiling additional functional aspects, particularly in scenarios where biotic and abiotic stress responses intersect. Thus, we focused on the bHLH genes and explored the gene function based on large scales of expression profiles from RNA-seq data sets including Xoo infection and temperature stresses. Through this rigorous exploration, we were able to identify putative core bHLH genes that not only participate in distinct stress responses but may also serve as crucial mediators in the crosstalk between Xoo infection and temperature stress. The findings from this work provide a significant step forward in unraveling the complex network of stress response and signal transduction in rice.

The combined stresses from various environmental and pathogenic factors pose a substantial challenge to crop survival and productivity, necessitating in-depth comprehension of the underlying molecular mechanisms. Recent studies have shown that the response of plants to these combinations is unique and cannot be directly extrapolated from the response to individual stresses. The complexity of plant responses to stress combinations is largely controlled by different and sometimes opposing signaling pathways that may interact and inhibit each other, making it challenging to predict the impact on plant growth and physiological traits (Atkinson and Urwin, 2012; Suzuki et al., 2014). The basic helix-loop-helix (bHLH) superfamily, a diverse set of transcription factors, plays a pivotal role in mediating plant responses to these combined stresses (Heim, 2003; Pires and Dolan, 2010; Feller et al., 2011). Considering the complexity of Xoo infection in rice causing bacterial leaf blight (Niño-Liu et al., 2006). This bacterium employs various virulence factors, leading to a complex infection process (Büttner and Bonas, 2010). The plant immune system provides an intricate line of defense against such pathogens, contributing to the complexity of the interaction (Jones and Dangl, 2006). Given the potential role of bHLH transcription factors in plant stress responses, we decided to integrative analysis of RNA-seq from different perspectives to identify functional bHLH genes. In this study, we have incorporated three distinct RNA-seq datasets that provide a comprehensive perspective on the interaction between Xoo and rice. The datasets include scenarios with Xoo strains that are either without TAL effectors, or have the avrXa23 gene disrupted, and injected into CBB23 (JG30 + Xa23) cultivar, and its background cultivar JG30. TAL effectors, injected into the plant cells by Xoo, bind to specific plant genomic promoters, modulating gene expression in a way that often enhances conditions for bacterial prosperity and augments disease progression (Boch and Bonas, 2010). Contrastingly, plant R (resistance) genes play an instrumental role in the immune system by identifying and responding to specific pathogens or disease-causing agents (Jones and Dangl, 2006). Thus, by integrating these datasets, our analysis covers both pathogen-driven and host-mediated aspects of the bacterial blight (BB) disease, allowing for a deeper understanding of gene expression dynamics over time.

From the individual dataset, we identified 6, 10, 5 bHLH genes differentially regulated between treatment and control rice tissues ( Figures 3 - 5 ). In general, these genes were induced in the relatively later phases of the infection experiments, most during 24–48 hp. Upon pathogen exposure, plants rapidly initiate a multi-tiered defense response to prevent pathogen invasion and propagation. The first line of defense, known as pathogen-associated molecular pattern-triggered immunity (PTI), is launched within minutes to hours of pathogen recognition, facilitated by pattern recognition receptors (PRRs) such as FLS2 and EFR that perceive specific molecular patterns on the pathogen surface fun (Boutrot and Zipfel, 2017). Signal perception and transduction trigger the activation of multiple downstream processes, including calcium influx, production of reactive oxygen species (ROS), activation of mitogen-activated protein kinase (MAPK) cascades, and induction of defense-related genes (Bigeard et al., 2015). Central to these early responses is the involvement of several transcription factors (TFs) that regulate the expression of defense-related genes. For example, WRKY33, a member of the WRKY family, is activated in the early stages of pathogen infection, mediating defense responses against necrotrophic pathogens in Arabidopsis through modulating the biosynthesis of phytoalexins and ROS homeostasis (Birkenbihl et al., 2012). Similarly, members of the MYB, bZIP, and NAC TF families, such as MYB44, bZIP10, NAC019, NAC055, and NAC072, are also rapidly induced upon infection, regulating various aspects of the defense response including stomatal closure, immune responses, cell death, and basal defense (Kaminaka et al., 2006; Jensen et al., 2008; Jung et al., 2008). Thus, we deduced that the differentially regulated OsbHLH may involve a later phase of the infection defense system, especially for the cross-validated Os04g0301500 and Os04g0489600. These could include genes involved in hormone biosynthesis, signaling, and response, as well as genes involved in the synthesis of defensive compounds, cell wall reinforcement, and programmed cell death (Jones and Dangl, 2006).

To further verify the potent function of Os04g0301500 and Os04g0489600 involved in stress response, we examine transcriptomic data sourced from a near-isogenic line (NIL) IRBB67, which carries both Xa4 and Xa7 and its parental variety, IR24, under combined Xoo infection and temperature conditions. Interestingly, we observed no significant differential expression except for a notable temperature-induced down-regulation of Os04g0301500 in IRBB67. It’s known that Xa7 imparts a durable and broad-spectrum resistance against bacterial blight disease in rice, showing enhanced efficacy under high-temperature conditions, while the effectiveness of other R genes decreases (Webb et al., 2010; Chen et al., 2021). Temperature improved the tolerance of Xoo in IRBB67. This could potentially clarify the observed downregulation of Os04g0301500, which we previously reported as a likely negative regulator, enhancing Xoo tolerance. Of note, Xa4 is present in almost all commercial indica hybrid rice varieties in China, while Xa3/Xa26 is distributed widely in both indica and japonica varieties (Hu et al., 2017; Deng et al., 2018). Encoded by Xa4 is a cell wall-associated kinase that confers race-specific resistance to Xoo throughout the rice growth stages by strengthening the cell wall, thereby preventing Xoo invasion (Sun et al., 2003; Afzal et al., 2008; Hu et al., 2017). However, it is plausible that these R genes do not interact with the bHLH genes. As for Xa23, the unique executor R gene, AvrXa23, has been discovered to be highly conserved across all tested Xoo isolates (Wang et al., 2014). This conservation might augment the virulence of Xoo, facilitating infection and proliferation within host plants. Furthermore, the widespread occurrence of AvrXa23 among natural Xoo strains might underpin the broad-spectrum resistance offered by Xa23 (Jiang et al., 2020). RNA-seq analysis corroborated the induction and suppressive capability of Xa23 concerning Os04g0301500 ( Figure 4 ). In summary, our findings suggest that Os04g0301500 may play a significant role in Xoo tolerance and thermal stress response in rice plants.

At the molecular level, the convergence of biotic and abiotic stress responses often involves hormonal cross-talk. Stress hormones like JA, salicylic acid (SA), and abscisic acid (ABA) regulate various aspects of plant defense and abiotic stress tolerance, and many TFs, including bHLH and WRKY proteins, are known to interact with these hormonal signaling pathways (Pieterse et al., 2009). One of the most well-documented examples of this phenomenon involves the SA-dependent pathway, typically activated in response to biotrophic and hemibiotrophic pathogens. Activation of R genes by these types of pathogens often leads to an increase in SA synthesis, which in turn triggers the expression of pathogenesis-related (PR) genes, contributing to localized and systemic acquired resistance (SAR) (Vlot et al., 2009). In rice, the bHLH transcription factor RERJ1 also reported involvement in wounding and drought response (Kiribuchi et al., 2005). To extend the understanding of Os04g0301500 in stress response, transcriptome data of O. sativa subjected to chilling (10 °C) and cold (4°C) stress were analyzed. Os04g0301500 only shows differentially regulated under cold between sensitive and tolerant rice cultivars. It is known that prolonged exposure to low non-freezing temperatures (around 4°C) can lead to cold acclimation, which increases freezing tolerance, and this involves the activation of CBF/DREB TFs. However the exact response of these TFs to a more moderate cold stress (such as 10°C) can depend on various factors including the plant species, the duration of the stress, and the plant’s developmental stage (Chinnusamy et al., 2007). As a result, we concluded that Os04g0301500, in addition to Xoo resistance and heat stress, may be implicated in cold acclimation ( Figure 8 ).

In this study, our results suggest that the transcription factor Os04g0301500 acts as a negative regulator of stress tolerance in response to bacterial blight (Xoo), as well as cold and heat stress in rice. We found that the expression of Os04g0301500 was not significantly influenced by the resistance gene Xa4, but appeared to be modulated in coordination with Xa7 under increasing temperature conditions, which are known to enhance tolerance to Xoo. Our data also revealed that Xa4 is indirectly regulated by the resistance gene Xa23, through effector-triggered immunity and associated defense mechanisms. It’s well established that plant defense responses against pathogens involve a complex network of signal transduction pathways that integrate both biotic and abiotic stress signals to modulate plant immune responses. In this context, it appears that Os04g0301500 might serve as a crucial node in this network, linking pathogen defense mechanisms with responses to abiotic stresses through hormonal signaling pathways. The precise mechanism through which Os04g0301500 coordinates these responses remains to be elucidated, but it’s plausible that it might be involved in modulating the activity of hormonal signaling pathways in response to stress. For instance, ethylene, salicylic acid, and jasmonic acid are known to play critical roles in coordinating plant responses to both biotic and abiotic stresses. Os04g0301500 could potentially influence the balance of these hormones, thereby fine-tuning the plant’s overall response to environmental stress. In conclusion, our findings highlight the potential role of Os04g0301500 in integrating biotic and abiotic stress responses in rice, potentially serving as a key regulator of plant resilience under changing environmental conditions. Future research is warranted to further elucidate the precise role and underlying mechanisms of Os04g0301500 in stress response modulation.

Publicly available datasets were analyzed in this study. This data can be found here: https://www.ncbi.nlm.nih.gov/sra (PRJNA482466, PRJNA525987, PRJNA433094, PRJNA314700, PRJNA610422 and PRJEB22031), https://rice-genome-hub.southgreen.fr/node/143/621.

ZQ: Writing – original draft. TR: Data curation, Software, Writing – review & editing. YZ: Data curation, Writing – review & editing. SS: Data curation, Writing – review & editing, Formal Analysis. XY: Formal Analysis, Writing – review & editing. DH: Formal Analysis, Writing – review & editing. TW: Writing – review & editing, Project administration. WF: Writing – review & editing, Project administration, Data curation, Formal Analysis.