cChinese strong-flavored liquor (CSFL) accounts for more than 70% of all Chinese liquor production. Microbes in pit mud play key roles in the fermentation cellar for the CSFL production. However, microbial diversity, community structure, and cellar-agerelated changes in pit mud are poorly understood. Here, we investigated the prokaryotic community structure and diversity in pit-mud samples with different cellar ages (1, 10, 25, and 50 years) using the pyrosequencing technique. Results indicated that prokaryotic diversity increased with cellar age until the age reached 25 years and that prokaryotic community structure changed significantly between three cellar ages (1, 10, and 25 years). Significant correlations between prokaryotic communities and environmental variables (pH, NH 4 ؉ , lactic acid, butyric acid, and caproic acid) were observed. Overall, our study results suggested that the long-term brewing operation shapes unique prokaryotic community structure and diversity as well as pit-mud chemistry. We have proposed a three-phase model to characterize the changes of pit-mud prokaryotic communities. C hinese strong-flavored liquor (CSFL), also called "Luzhou flavor liquor," accounts for more than 70% of Chinese liquor production (1). It is produced by the unique and traditional Chinese solid-state fermentation technique, which has a history of several thousand years. In brief, a cellar is constructed by digging a rectangular soil pit in which the entire inner wall is covered with precultured pit mud. The precultured pit mud is usually prepared by mixing aged pit mud (as an inoculum), fresh common soil, and water and incubating the mixture for about a year in an anaerobic cellar before use. The raw materials for the fermentation, including wheat, sorghum, and corn, are mixed, crushed, and distilled by steaming. The steamed raw material is supplied with 2% to 3% (wt/wt) Daqu-starter, which mainly includes mold and yeast, and placed into the cellar. The cellar is sealed with common mud, and fermentation is allowed to proceed for 60 days. Fermented material is then taken out of the cellar and distilled to make Chinese liquor. The process described above is periodically repeated after new fermentation materials are supplied.Microbes in the pit mud produce various flavor components such as butyric acid, caproic acid, and ethyl caproate. In particular, ethyl caproate is recognized as a key component affecting the CSFL flavor and quality. In general, CSFL quality improves with increasing cellar age. High-quality liquor is produced only in old cellars, which are maintained at least for 20 years by continuous use (2, 3). In particular, some long-aged cellars have been used for several hundred years without interruption, and well-known CSFLs such as Wuliangye, Jiannanchun, and Luzhoulaojiao are brewed in such long-aged cellars (1, 4). High CSFL quality is attributed to the maturing process of pit mud, which results in a well-balanced microbial community structure and diversity in the pit mud to produce distinctive flavo...

miR169 is a conserved microRNA (miRNA) family involved in plant development and stress-induced responses. However, how miR169 functions in rice immunity remains unclear. Here, we show that miR169 acts as a negative regulator in rice immunity against the blast fungus Magnaporthe oryzae by repressing the expression of nuclear factor Y-A (NF-YA) genes. The accumulation of miR169 was significantly increased in a susceptible accession but slightly fluctuated in a resistant accession upon M. oryzae infection. Consistently, the transgenic lines overexpressing miR169a became hyper-susceptible to different M. oryzae strains associated with reduced expression of defense-related genes and lack of hydrogen peroxide accumulation at the infection site. Consequently, the expression of its target genes, the NF-YA family members, was down-regulated by the overexpression of miR169a at either transcriptional or translational level. On the contrary, overexpression of a target mimicry that acts as a sponge to trap miR169a led to enhanced resistance to M. oryzae. In addition, three of miR169’s target genes were also differentially up-regulated in the resistant accession upon M. oryzae infection. Taken together, our data indicate that miR169 negatively regulates rice immunity against M. oryzae by differentially repressing its target genes and provide the potential to engineer rice blast resistance via a miRNA.

Rice false smut has emerged as a serious grain disease in rice production worldwide. The disease is characterized by the transformation of individual rice florets into false smut balls, which is caused by the fungal pathogen Ustilaginoidea virens. To date, little is known about the host factors required for false smut ball formation by U. virens. In this study, we identified histological determinants for the formation of false smut balls by inoculating U. virens into rice floral mutants defective with respect to individual floral parts. The results showed that U. virens could form mature false smut balls in rice floral mutants with defective pistils, but failed to develop false smut balls in the superwoman mutant lacking stamens, identifying that U. virens requires rice stamens to complete its infection cycle. Comparative transcriptome analysis indicated a list of candidate host genes that may facilitate nutrient acquisition by U. virens from the rice stamens, such as SWEET11, SWEET14 and SUT5, and genes involved in the biosynthesis of trehalose and raffinose family sugars. These data pinpoint rice stamens as the key target organ of U. virens infection and provide a valuable starting point for dissecting the molecular mechanism of false smut ball formation. Received

ORCID IDs: 0000-0002-6747-4302 (J.F.); 0000-0003-4066-4875 (W.-M.W.).Circular RNAs (circRNAs) play roles in various biological processes, but their functions in the rice (Oryza sativa) response to Magnaporthe oryzae remain elusive. Here, we demonstrate that circRNAs are involved in the rice-M. oryzae interaction using comparative circRNA-sequencing and transgenic approaches. We identified 2932 high-confidence circRNAs from young leaves of the blast-resistant accession International Rice Blast Line Pyricularia-Kanto51-m-Tsuyuake (IR25) and the blast-susceptible accession Lijiangxin Tuan Heigu (LTH) under M. oryzae-infected or uninfected conditions; 636 were detected specifically upon M. oryzae infection. The circRNAs in IR25 were significantly more diverse than those in LTH, especially under M. oryzae infection. Particularly, the number of circRNAs generated per parent gene was much higher in IR25 than in LTH and increased in IR25 but decreased in LTH upon M. oryzae infection. The higher diversity of circRNAs in IR25 was further associated with more frequent 39 and 59 alternative back-splicing and usage of complex splice sites. Moreover, a subset of circRNAs was differentially responsive to M. oryzae in IR25 and LTH. We further confirmed that circR5g05160 promotes rice immunity against M. oryzae. Therefore, our data indicate that circRNA diversity is associated with different responses to M. oryzae infection in rice and provide a starting point to investigate a new layer of regulation in the rice-M. oryzae interaction.