Physiological ER stress caused by amylase production induces regulated Ire1-dependent mRNA decay in Aspergillus oryzae

Regulated Ire1-dependent decay (RIDD) is a feedback mechanism in which the endoribonuclease Ire1 cleaves endoplasmic reticulum (ER)-localized mRNAs encoding secretory and membrane proteins in eukaryotic cells under ER stress. RIDD is artificially induced by chemicals that generate ER stress; however, its importance under physiological conditions remains unclear. Here, we demonstrate the occurrence of RIDD in filamentous fungus using Aspergillus oryzae as a model, which secretes copious amounts of amylases. α-Amylase mRNA was rapidly degraded by IreA, an Ire1 ortholog, depending on its ER-associated translation when mycelia were treated with dithiothreitol, an ER-stress inducer. The mRNA encoding maltose permease MalP, a prerequisite for the induction of amylolytic genes, was also identified as an RIDD target. Importantly, RIDD of malP mRNA is triggered by inducing amylase production without any artificial ER stress inducer. Our data provide the evidence that RIDD occurs in eukaryotic microorganisms under physiological ER stress.

In this manuscript, the authors insist that Regulated Ire1-dependent decay (RIDD) occurs under physiological ER stress in Aspergillus oryzae.To support this, the authors treated DTT and checked mRNA levels of amyA/B/C.The authors also used ireA, hacA, and ski2 mutant strains and examined amyA/B/C and malP expression.It might be useful for understand the ER stress response.There are some comments.
-The authors can detect amyB in Figure 2.Then, it seems possible to check the expression of the amyA, amyB, and amyC genes.-It's much better to focus on the amyB expression.
-The amyB protein production or enzyme activity should be examined.
-The cleavage pattern of amyA/B/C in Figures 1 and 3 can be provided.
-The summary figure can be useful for understand this manuscript.
Reviewer #3 (Remarks to the Author): In this manuscript, the authors found that the mechanism of regulatory Ire1-dependent decay (RIDD) exists in filamentous fungi, Aspergillus oryzae, and they also found the physiological conditions under which RIDD occurs.The authors found that RIDD of malP mRNA is strongly induced by the production of amylolytic enzymes, rather than by chemical treatments that induce an endoplasmic reticulum stress response.These results are very interesting because they show that, in Aspergillus oryzae, both UPR and RIDD are induced by physiological endoplasmic reticulum stress.Although the experiments are carefully conducted and the paper is carefully written, some parts are a bit difficult to read.The following points should be further addressed or modified.
Major points Fig. 3: The authors should compare the ireA mutant strain with the hacA mutant strain.Fig. 2 and Fig. 4: The frame shift mutant applied to amyB should be analyzed with malP in Fig. 4.
3) Figure 4b: It is hard to understand the decrease in the short fragment of amyA/B/C mRNA.How about quantification?4) Page 4, line 24 with mutation→with a mutation 5) Page 6, line 21-22.The authors mentioned "After adding DTT, the mRNA level of amyB-FS was found to be not much lower than that of the intact amyB (Fig. 2c)".This sentence seems a bit confusing.For example: "Decrease in the mRNA level of amyB-FS was not observed compared to that of the intact amyB upon DTT treatment".6) Discussion page 11, lines 23-25.The authors mentioned "The transcript of the frameshift mutant of amyB (amyB-FS) gradually decreased upon DTT treatment, although its rate of decrease was slower than that of the wild-type amyB mRNA".This result should be mentioned in the Results section.7) Discussion, p. 12, lines 6-8.The authors mentioned "As ireA-repressed cells expressing hacAi suppressed the reduction in the amyA/B/C mRNA levels treated with DTT, we speculated that RIDD and RESS might be regulated by different pathways."This result should be mentioned in the Results section.8) Discussion, page 12, line 25 -page 13, line 1.The authors mentioned "Dysfunction of RIDD due to loss of the Ski complex caused severe growth inhibition in maltose medium."They did not show the data which indicate that Dysfunction of RIDD caused severe growth.We thank the reviewer for very positive comments.We are very happy to receive such honorary comments.

Reviewer #2 (Remarks to the Author):
In this manuscript, the authors insist that Regulated Ire1-dependent decay (RIDD) occurs under physiological ER stress in Aspergillus oryzae.To support this, the authors treated DTT and checked mRNA levels of amyA/B/C.The authors also used ireA, hacA, and ski2 mutant strains and examined amyA/B/C and malP expression.It might be useful for understand the ER stress response.There are some comments.
We thank the reviewer for helpful comments.We have addressed all comments.
-The authors can detect amyB in Figure 2.Then, it seems possible to check the expression of the amyA, amyB, and amyC genes.
As described in page 5, lines 17-18, the sequences of amyA, amyB, and amyC are almost identical, so these genes cannot be individually detected.In Figure 2, we used an A. oryzae strain lacking all three copies of the intrinsic α-amylase genes (∆amyA/B/C) as a host for expression of wild type or mutant amyB (amyB-FS) genes to specifically detect these amyB transcripts.

-It's much better to focus on the amyB expression.
The ∆amyA/B/C strain was used only for expression of amyB driven by actA promoter (Fig. 1, right panel) or amyB-FS (Fig. 2C).Therefore, it is not possible to detect native amyB mRNA as distinct from amyA or amyC mRNAs.
-The amyB protein production or enzyme activity should be examined.
When A. oryzae cells are treated with DTT, the newly synthesized secreted proteins do not fold properly and may be degraded by ER-associated degradation.In addition, pre-existing ones misfold due to the reduction of disulfide bonds.These may lead to difficulties in detecting the α-amylase production or its enzyme activity.
-The cleavage pattern of amyA/B/C in Figures 1 and 3 can be provided.
As described in page 7, lines 18-20, the short fragments of the amyA/B/C mRNAs resulting from cleavage by IreA are rapidly degraded by the 3′ to 5′ mRNA degradation pathway owing to lack of a poly(A) tail.Therefore, they can only be clearly detected in a disruption mutant strain of Aoski2 involving in the 3′ to 5′ mRNA degradation pathway.Since A. oryzae strains used for experiments in Figure 1, 3A, and 3B are not deficient in Aoski2, the cleavage products of amyA/B/C mRNA are not detectable.
-The summary figure can be useful for understand this manuscript.
In accordance with this reviewers' comment, we show the summary figure as new Fig. 6.

Reviewer # 1 (
Remarks to the Author):In the manuscript by Tanaka et al the authors investigate the mechanism underlying the phenomenon of reduced transcript levels of genes encoding secreted proteins under ER stress conditions in Aspergillus oryzae.Using the constitutive actA promoter, the authors demonstrate that reduced transcript levels of amyA/B/C and glaA after induced ER stress are not (only) based on the previously coined term RESS (repression under secretion stress).They furthermore demonstrate convincingly the requirement of ER targeting and the presence of IreA for degradation of the amyB and malP mRNA.Reduced degradation in the ∆ski2 background further suggests that the amyA/B/C and malP mRNAs are degraded via RIDD (regulated Ire1-dependent decay) and the 3'-5'exosomal degradation pathway.Importantly, RIDD of amyA/B/C and of malP mRNAs is induced under physiological conditions.Taken together, the authors reveal that RIDD, which was previously thought to occur only in higher eukaryotes and fungi without a functional Ire1/Hac1 pathway (S. pombe and Candida glabrata), functions in Aspergillus oryzae (and likely also other Aspergilli) to reduce the folding demand in/of the ER.This paper is very well and clearly written.Intro/abstract and discussion are on point.It has not happened very often to me that I do not have any major or minor points to be addressed.Congratulations to this wonderful piece of work adding truly novel and important insight, expanding the knowledge on the unfolded protein response in fungi and in general.

Fig. 6 .
Fig. 6.Schematic diagram showing the response to physiological ER stress induced by amylolytic enzyme production.(a) Expression of amylolytic genes is induced by maltose uptake through MalP, and the unfolded proteins resulting from the synthesis of nascent polypeptides from their mRNAs induce ER stress.(b) The resulting ER stress activates IreA, leading to HacA activation and RIDD.Thereafter, RIDD and the HacA-mediated UPR and RESS alleviate ER stress.In addition to RIDD and RESS, carbon catabolite repression (CCR) and MalP endocytosis induced by glucose generated from the maltose hydrolysis also contribute to the decrease in amyA/B/C and malP mRNAs.The correctly folded protein is represented by the three-dimensional structural image of α-amylase (PDB ID: 6TAA).The red arrows indicate the activation of transcription factors and CCR.