Function of PoLAE2, a laeA homolog, in appressorium formation and cAMP signal transduction in Pyricularia oryzae

ABSTRACT

A novel homolog of laeA, a global regulatory gene in filamentous fungi, was identified from Pyricularia oryzae. A deletion mutant of the homolog (PoLAE2) exhibited lowered intracellular cAMP levels, and decreased appressorium formation on non-host surface; the decrease was recovered using exogenous cAMP and IBMX, indicating that PoLAE2 deletion affected the cAMP signaling pathway.

KEYWORDS:

• LaeA
• appressorium formation
• signal transduction
• cAMP

Rice (Oryza sativa) is a crucial crop for humans and livestock. In the past, rice cultivation was interrupted due to persistent infection of the crop with plant pathogens. Rice blast fungus is one of the most destructive fungal pathogens that cause rice blast disease. The fungus is distributed in and has devastated many regions where the rice is cultivated worldwide. It is estimated that each year rice blast causes harvest losses of 10–30% of the global rice yield [1]. This problem affects farmers’ income and global food security. Therefore, understanding the disease mechanism is a necessary strategy to control the disease for sustainable food supply.

Many plant pathogenic fungi have evolved the capacity to breach the intact cuticles of their plant hosts by employing specialized structures called appressoria [2]. Appressorium formation in Pyricularia oryzae is regulated by cell cycle progression. Once formed, the appressorium matures and generates turgor pressure by accumulating high concentrations of compatible solutes, such as glycerol [3]. Then, penetration peg appears, and invasive hyphae grow biotrophically in host cells [4]. In rice blast fungus, appressorium formation is triggered by physical and chemical signals from the host surface. Moreover, several genetic studies have indicated a linkage of appressorium formation with signal transduction pathways, including the cyclic AMP (cAMP) signaling pathway.

laeA is a regulatory gene in ascomycete fungi that is required for cell signaling to control the expression of many secondary metabolites and for morphogenesis [5,6]. The gene is well conserved in numerous fungi, suggesting that it has important evolutionary functions in fungal physiology [7]. laeA of many plant pathogenic fungi have been reported to encode a nuclear protein that is required for the expression of secondary metabolite genes [8]. LaeA serves as a regulator of these processes and has been shown to be involved in controlling the switch between sexual and asexual development in various ascomycetes [9]. Recently, Saha et al. [10] revealed the role of MoLAEA in the secondary metabolism of P. oryzae. However, a similarity search of the amino acid sequence database using the Aspergillus fumigatus LaeA (AY422723) hit another homolog in P. oryzae (MGG_08161). The amino acid sequence of MGG_08161 showed homology to that of A. fumigatus LaeA (33%) and MoLAEA (38%) (Supplementary figure S1). The gene was named PoLAE2, and its deletion mutant and complementation strains were constructed in this study to investigate its function.

In order to delete PoLAE2, the pDESTR system [11] was utilized. First, an inverse PCR was performed using two primers (5′-CACCCCGCCTGAAATCTTGT-3′ and 5′-GTGCGTAGTAATCGTGG-3′) with SmaI-digested, self-ligated genomic DNA, and then, a disruption vector was constructed using pBARST [11]. For the construction of the PoLAE2 complementation vector, two primers (5′-CACCCTCCCTGATGTCGTGGTTTT-3′ and 5′-TGGGCTCTTCAGGATATTGG-3′) were used to amplify the DNA containing the putative promoter and PoLAE2. The fragment was also ligated to pBLASTR [11]. Transformation of P. oryzae Ina86-137Δlig4 (for the disruption of PoLAE2) and Ina86-137Δlig4Δlae2 (for the complementation) was performed according to protoplast-polyethylene glycol method as described previously [11,12]. The formation of the disruption mutant and complementation of the mutant were confirmed by Southern hybridization [13] (Supplementary figure S2).

The mycelial growth rates of Ina86-137 (wild type), Ina86-137Δlig4Δlae2 (PoLAE2 deletion mutant), and two complementation strains (T9 and T11) were not significantly different (data not shown). To determine the function of PoLAE2 in appressorium formation on non-host plants and rice, inoculum of wild type, Ina86-137Δlig4Δlae2, T9, and T11 were prepared at the concentration of 10⁴ conidia/mL for onion epidermis assay and intact leaf sheath assay and 10⁵ conidia/mL for the spray inoculation assay. The onion epidermis assay [14,15] was used to analyze appressorium formation. More than 80% appressorium formation was detected on onion epidermis after it was inoculated with wild type and each complementation strain. Conversely, the formation of the appressorium by PoLAE2 deletion mutant was significantly decreased to less than 20% (Figure 1). These results indicate that the deletion of PoLAE2 affected appressoria formation.

Figure 1. Formation of appressorium of PoLAE2 deletion mutant on onion epidermis. (a) Percentage of appressorium formation without treatment, with 10 mM exogenous cyclic AMP (cAMP), or with 2.5 mM 3-isobutyl-1-methylxanthine (IBMX). (b) Microscopic image of onion epidermis. WT, Ina86-137 (wild type); MT, Ina86-137Δlig4Δlae2 (PoLAE2 deletion mutant); T9 and T11, complementation strains. Scale bars = 50 µm.

To investigate the pathogenicity of Ina86-137Δlig4Δlae2 in rice, a spray inoculation assay [12] and an intact leaf sheath assay [16] were conducted to clarify the pathogenicity of rice blast fungus on the Oryza sativa cultivar Shin-2. The results indicated that all rice samples that were sprayed with PoLAE2 deletion mutant and complementation strains (T9 and T11) showed the development of necrotic lesions on their leaves, which were similar to those formed by the wild type fungus (Figure 2(a)). Furthermore, the results of the intact rice leaf sheath assay confirmed appressorium formation on the rice leaf sheath by each fungal strain (Figure 2(b)). These results indicate that the lack of PoLAE2 might not affect appressorium formation on rice. However, it is required for appressorium formation on non-host surface.

Figure 2. Pathogenicity of PoLAE2 deletion mutant in rice. (a) Spray inoculation and (b) intact leaf sheath inoculation of susceptible Shin-2-rice with conidia of Ina86-137 (wild type, WT), Ina86-137Δlig4Δlae2 (PoLAE2 deletion mutant, MT), and complementation strains (T9 and T11). Scale bars = 50 µm.

In order to determine the role of PoLAE2 in cAMP signal transduction in appressorium development, 10 mM exogenous cAMP or 2.5 mM 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor) was applied to conidia suspension and appressorium formation on onion epidermis was analyzed as described in a previous study [17,18]. cAMP treatment resulted in the recovery of appressorium formation in the PoLAE2 deletion mutant by up to 59%. Furthermore, we found that appressorium formation could be increased to 70% upon induction of PoLAE2 deletion mutant with IBMX (Figure 1(ab)). In addition, PoLAE2 deletion mutant also showed decreased appressorium formation on the hydrophobic surface of gelbond film (Lonza, Rockland, ME), and the recovery by external addition of IBMX (data not shown).

These results suggested that PoLAE2 deletion seems to affect cAMP signaling by lowering cAMP concentration. To examine the effect of PoLAE2 on intracellular cAMP levels in rice blast fungus, each fungal strain was cultured in 2YEG broth (yeast extract 2 g/L and glucose 10 g/L) at 27°C for 5 days, and intracellular cAMP was extracted from each fungal mycelium after lyophilization according to the procedure described by Liu et al. [19]. The cAMP levels were quantified by HPLC as described previously [20]. The results showed that intracellular cAMP levels were significantly lower in PoLAE2 deletion mutant than in wild type and complementation strains (p < 0.05, Figure 3). Moreover, in order to elucidate the relationship between PoLAE2 and two crucial genes that are concerned with the maintenance of intracellular levels of cAMP, MAC1 [21] and PDEH [22], in rice blast fungus, reverse transcription-polymerase chain reaction (RT-PCR) was conducted using RNA extracted from appressorium of each fungal strain. The results showed that there was no significant difference in the transcription of these genes among the strains tested (Supplementary figure S3). Therefore, PoLAE2 might not be directly involved in the regulation of MAC1 and PDEH at the transcriptional level.

Figure 3. Quantification of cAMP in fungal mycelium. cAMP was extracted from the mycelia of Ina86-137 (wild type, WT), Ina86-137 Δlig4Δlae2 (PoLAE2 deletion mutant, MT), and complementation strains (T9 and T11).

These results revealed two things. One is that PoLAE2 deletion affected cAMP signaling by decreasing intracellular cAMP concentration. Bok and Keller [8] revealed that cAMP signaling negatively regulates LaeA, but no previous studies on fungal LaeA homolog have reported on regulation of cAMP signaling downstream of LaeA. Recently, Yanyong et al. [23] conducted a proteomics analysis of Aspergillus flavus and found that the deletion of laeA in A. flavus resulted in the upregulation of proteins or enzymes involved in chromatin remodeling and modification. The relationship between chromatin remodeling and appressorium formation has been pointed out in P. oryzae [24,25]. It might be possible that PoLAE2 is related to the chromatin remodeling, but further omics (transcriptomics, proteomics, etc.) investigation on PoLAE2 deletion mutant will be required for the elucidation of the regulation mechanism of cAMP signaling by PoLAE2.

Another novel finding is that homeostasis of intracellular cAMP levels is required for proper appressorium formation, and PoLAE2 plays a role in homeostasis. As discussed above, cAMP signaling and PoLAE2 can make a feedback loop, which might be a concern in cAMP homeostasis. Choi and Dean [19] showed that MAC1 deletion mutant of rice blast fungus was unable to form appressoria on an inductive surface, and appressorium formation was restored in the presence of exogenous cAMP derivatives. Conversely, Ramanujam and Naqvi [20] found that loss of PDEH led to increased accumulation of intracellular cAMP during infectious growth and significantly accelerated appressorium formation. These studies used deletion mutants for the genes involved in cAMP production and decomposition, and therefore, the results were based on an all-or-nothing effect. In addition, in this study, we found that elevated cAMP levels may concern with appressorium formation on the non-host surface. An unknown host factor might compensate for the lowered level of cAMP during appressorium formation on the host surface.

In conclusion, PoLAE2 plays a role in the accumulation of cAMP, which is sufficient for appressorium formation on non-host surfaces. Additional research is required to elucidate the linkage between PoLAE2 and cAMP signaling because the linkage should be complicated, as suggested by the results of RT-PCR of MAC1 and PDEH. However, this is the first report to reveal the regulation of cAMP signaling by the LaeA homolog in filamentous fungi, and additional research on this global regulator will reveal its function other than secondary metabolism.

Author contributions

TS, AA, and PP designed the research. PP, VK, JA, WS, and AA performed the experiments. PP, AA, and TS wrote the manuscript. All authors have reviewed and approved the final manuscript.

Compliance with ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Acknowledgments

This study was supported by a scholarship from Rajamangala University of Technology, Thanyaburi, Thailand, awarded to PP for studying at Hokkaido University, Japan. The authors would like to thank Editage (www.editage.com) for English language editing.

Disclosure statement

The authors declare that they have no conflicts of interest.

Supplementary material

Supplemental data for this article can be accessed here.