Ustilaginoidea virens: A Fungus Infects Rice Flower and Threats World Rice Production

Qiu Jiehua, Meng Shuai, Deng Yizhen, Huang Shiwen, Kou Yanjun

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: A Fungus Infects Rice Flower and Threats World Rice Production

Qiu Jiehua1, Meng Shuai1, Deng Yizhen2, Huang Shiwen1, Kou Yanjun1

(State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China; Guangdong Province Key Laboratory of Microbial Signals and Disease Control / Integrative Microbiology Research Centre, South China Agricultural University, Guangzhou 510642, China)

Rice false smut disease, which is caused by the fungus, is currently one of the most devastating rice fungal diseases in the world. Rice false smut disease not only causes severe yield loss and grain quality reduction, but also threatens food safety due to its production of mycotoxins. In this review, the most recent progresses regarding the life cycle, infection processes, genome and genetic diversity, pathogenic gene and disease resistance in rice were summarized in order to provide theoretical basis for the control of. We also proposed some future directions and key questions that need to be addressed for a better understanding of the molecular mechanism that leads to rice false smut disease and the prospects for sustainable control of rice false smut.

; rice false smut disease; life cycle; infection process; pathogenesis gene; disease resistance

(Cook) Takahshi (teleomorph form:) is an ascomycete fungal pathogen which causes rice false smut disease. Rice false smut disease once occurred sporadically in rice planting areas. However, it is one of the most devastating rice fungal diseases due to the widespread use of high- yielding cultivars and hybrids, superfluous applications of chemical fertilizers and climate change. In China, it has been estimated to have occurred in approximately 2.4 million hectares per year between 2015 and 2017 (www.natesc.org.cn/sites/MainSite/). It is particularly prevalent in Anhui, Hunan, Hubei, Jiangsu, Jiangxi and Zhejiang provinces of China (Lu et al, 2018). In addition, this disease has also been recently reported with increasing frequency in rice growing areas worldwide (Guo et al, 2012; Ladhalakshmi et al, 2012; Jecmen and Tebeest, 2015; Nessa et al, 2015; Kumagai et al, 2016). The typical symptom of rice false smut disease is the replacement of rice grains with false smut balls, which are at first yellowish orange to green in color, and finally turn a greenish-black (Fig. 1).

Rice false smut disease causes severe yield loss and reduces grain quality. It also contaminates rice grain and straw with mycotoxins, which raises great concerns for food and feed safety. Therefore, rice false smut disease andhave gained increased attention from researchers, breeders and farmers. In this review, the progress which has been recently made in the research ofthis unique pathological system was summarized, including the life cycle, infection processes and functional genomics of, along with the rice resistance to this disease, to highlight the molecular mechanism of the interactions of rice-.

Life cycle of U. virens

The life cycle ofinvolves both sexual and asexual stages (Zhang et al, 2014).exists in heterothallic form, and mating compatibility is determined by mating-type locus 1 (MAT1-1 andMAT1-2) (Yu J J et al, 2015). In the sexual cycle, sclerotia, which are induced by low temperatures, form on the surfaces of the rice false smut balls in late autumn (Fan et al, 2016). Under appropriate wetness, light and temperature conditions, the sclerotia germinate to differentiate the stroma, and then form asci with ascospores (Singh and Dubey, 1984; Yong et al, 2018a). The ascospores produce secondary conidia to contribute to primary infections of rice (Yong et al, 2018a). During the asexual cycle, thick-walled chlamydospores form on the surfaces of the false smut balls. The chlamydospores serve as important sources of inoculum between the seasons (Lu et al, 1994). As ascospores, the chlamydospores produce secondary conidia which result in rice false smut disease (Rush et al, 2000; Zhang et al, 2003). In addition to rice, alternative hosts such as weeds (,,and) may also be involved in the life cycle ofwith rare infections observed (Shetty and Shetty, 1985, 1987; Atia, 2004).

Fig. 1. Ustilaginoidea virens causes rice false smut disease.

Although increasing knowledge has been gained regarding the life cycle of, the debate continues as whether sclerotia or chlamydospores are the most important primary inoculum in the field, with final conclusion remaining elusive. It has been found that both the sclerotia and chlamydospores can survive over ten months in the laboratory conditions or field conditions (Lu et al, 1994; Wang, 1995). Therefore, both sclerotia and chlamydospores have the potential to be primary inoculum in the field. The results of recent studies have shown that sclerotia can be largely produced in many different geographical regions, including temperate and subtropical zones (Yong et al, 2018a). The sclerotia germinate to form ascospores under light condition after 2- to 5-month dormancy period. The ascospores are usually trapped in the rice-paddy fields before and after rice planting, which suggests that sclerotia can successfully overwinter and regularly produce ascospores (Yong et al, 2018a). Meanwhile, the chlamydospores are only trapped in the rice-paddy fields when the disease symptoms appeared. All of the aforementioned results supported the hypothesis that the sclerotia act as the primary inoculum of, rather than the chlamydospores(Yong et al, 2018a)

Infection processes of U. virens

Understanding the infection processes ofis critical in the study and management of rice false smut disease. The first question that must be addressed is the initial infection sites ofIt has been reported thatcan potentially infect coleoptiles during the seed germination stage, and also infect the seedling roots (Zheng et al, 2009; Andargie et al, 2015; Prakobsub and Ashizawa, 2017). However, the infections of the coleoptiles and roots may not spread to the spikelets and cause the characteristic symptom of rice false smut disease as no invasive hyphae have been observed beneath the pedicels or in the stems of naturally severely infected panicles (Tang et al, 2013; Yong et al, 2018b). Recently, some reports have suggested that the initial infections may occur in the rice pistils (Chao et al, 2014; Andargie et al, 2016). However, more studies have shown thatspecifically and initially infects the rice filaments before the rice flowers open (Tang et al, 2013; Hu et al, 2014; Zhang et al, 2014; Fan et al, 2015; Song et al, 2016). Collectively, through the natural observation (Shen, 2004), artificial inoculation assay and serial histological studies, it has been determined thattends to first infect the rice spikelets (mainly through the rice filaments) during the booting stage resulting in rice false smut disease. However, when and how the conidia enter the rice spikelets in nature remains unknown.

According to the current understanding ofinfection processes, under proper conditions, the conidia ofgerminate and form a large number of secondary conidia and hyphae on the surfaces of rice spikelets (Fan et al, 2014). Then, the extended hyphae enter the inner spaces of the spikelets through small gaps between the lemma and palea to infect the stamen filaments, and possibly the stigma or lodicules, without haustorium or appressorium, at approximately 4 dpi (days post inoculation) (Ashizawa et al, 2012; Li et al, 2013; Tang et al, 2013; Hu et al, 2014; Fan et al, 2015; Song et al, 2016). It has been noted that the invasive hyphae ofextend along the cell gaps of the filaments without penetration of the host cell walls by transmission electron microscope observation (Tang et al, 2013). The initiation ofinfection blocks the pollination process, and mimics the fertilization of the rice ovaries to hijack the rice nutrient supply (Fan et al, 2015; Song et al, 2016). This is the characteristic of the infection process ofcompared with other rice fungal pathogens(Talbot, 2003). At approximately 10 dpi, the hyphae have covered the stamen anthers, stigmas, and styles of the pistils, and begin to grow out of the spikelets. Finally, ball-like colonies, which are caused by infection of one or multipleisolates, are formed at 15 dpi (Yu M N et al, 2013). In the rice smut balls, the rice ovaries remain alive, indicating thatdoes not kill the host cell during its infection process, and is a biotrophic parasite (Tang et al, 2013).

Mycotoxins in U. virens

Rice false smut pathogen produces mycotoxins, including ustiloxins and ustilaginoidins. The ustiloxins contain a 13-membered cyclic core structure with a phenol ether linkage, including ustiloxins A, B, C, D, E, F and G in(Koiso et al, 1994, 1998; Wang et al, 2017; Lin et al, 2018). The ustiloxins ofinhibit the microtubule assembly and skeleton formation of eukaryotic cells (Koiso et al, 1994, 1998; Wang et al, 2017; Lin et al, 2018). Ustilaginoidins are a class of bis-naphtho-γ-pyrones, which have cytotoxic activities on cancer cells and inhibitory effects on the radicle elongation of rice seeds (Lu et al, 2015; Meng et al, 2015; Wang et al, 2016; Sun et al, 2017). To date, 26 ustilaginodins, namely ustilaginodins A, B, C, D, E, E1, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V and W, isochaetochromin B2, and 2,3-dihydroustilaginoidin T, have been identified in(Lu et al, 2015; Meng et al, 2015; Sun et al, 2016, 2017). Furthermore, several analytical methods have been established, such as high performance liquid chromatography (HPLC), liquid chromatograph-mass spectrometry (LC-MS), enzyme linked immunosorbent assay (ELISA), and lateral flow immunoassays (LFIA) for detecting or quantifying the mycotoxins of(Shan et al, 2012; Bian et al, 2015; Fu X X et al, 2015a, b, 2017; Cao et al, 2016). Although many mycotoxins have been successfully identified in, at least five aspects still require further exploration, including the activities and toxicities of each mycotoxin in humans or animals, the functions of mycotoxins during infection of, the differences of the mycotoxins in differentisolates, the molecule mechanism of mycotoxin synthesis inand new types of mycotoxins besides ustiloxins and ustilaginoidins.

Genome and genetics of U. virens

The 39.4 Mb draft genome sequences of(strain), including 8 426 predicted genes and approximately 25% repetitive sequences which are affected by repeated-induced point mutation, were available from 2014 (Zhang et al, 2014). Intriguingly,is evolutionarily close to the entomopathogenicspp. Several features of thegenome may provide cues into its ability to cause disease in rice. These features include an elaborate predicted secretome of up to 628 proteins, which may be highly expressed during the early infection stage (Zhang et al, 2014). In addition, thegenome displays reduced genes related to polysaccharide degradation, G-protein receptors and transporters, and secondary metabolism comparing with the majority of the other sequenced ascomycetes which may be associated with its biotrophic lifestyle. Furthermore, a predicted protein-protein interaction network was constructed based on the availability of the genome sequences and gene expression profiles duringinfection (Zhang et al, 2017). The aforementioned protein-protein interaction network database, which provides insights into protein functions of, is available at http://sunlab.cau.edu.cn/uvpid (Zhang et al, 2017).

shows significant genetic diversity in its isolates from different rice production areas due to geographical environments rather than rice cultivars (Pan et al, 2007; Li et al, 2013; Lu et al, 2013; Sun et al, 2013; Wang F et al, 2014; Wang W B et al, 2014b). Different geographical populations ofisolates display significant genetic differences, while isolates from different rice cultivars in the same areas, have higher genetic similarities (Sun et al, 2013). To date, the relationship between the pathogenicity and genetic diversity ofremains unclear.

Pathogenic genes of U. virens

Although thegenome has been sequenced, studies regarding the virulence factors and pathogenic genes are few due to the limitation of relative low frequency of the targeted gene deletion by homologous recombination, which is an important approach to study gene functions in plant pathogenic fungi, and the stability issues of artificial inoculations (Zheng et al, 2016). Five genes, namely,,,and,have been identified as the T-DNA insertion sites of the random T-DNA insertion mutants of, which are evident as defects in the sporulation, hypha growth, and/or pathogenesis (Yu J J et al, 2013; Huang et al, 2013; Yu M N et al, 2015; Wang et al, 2015; Bo et al, 2016). In addition, 17genes have been found to be up-regulated during the infection stage, which suggests that these genes may be also involved in the pathogenicity of(Yin et al, 2017). However, the functions of the aforementioned genes require further confirmation and investigation using targeted gene deletion or RNAi strategies in.

To date, only,,andgenes have been functionally characterized by generating knockout mutants or RNAi transformants (Lv et al, 2016; Zheng et al, 2016; Liang et al, 2018). The first is theHOG1 homologwhich plays a conservative role in regulation stress responses, hyphal growth, and possibly secondary metabolism (Zheng et al, 2016). Only onedeletion mutant was identified in more than 600-resistance transformants by Zheng et al (2016), which suggests that the frequency of homologous recombination and target gene replacement is much lower inthan(Talbot, 2003). Bothandhave been identified from a random T-DNA insertion mutant, and are required for sporulation, hypha growth and pathogenesis (Lv et al, 2016; Zheng et al, 2016; Zheng et al, 2017). Recently, the functions of, which involves in stress response and conidiation, have been characterized using the CRISPR-Cas9 system. Although the insertion of thegene may disrupt other gene and complementation analysis in the mutant background is difficult, the combination of CRISPR-Cas9 system and homologous recombination is an efficient approach for targeted gene deletion into date. This methodremoves a technical limitation of the analyses of gene function in. Therefore, by using this method, more pathogenic genes and related signaling pathways can potentially be elucidated in the near future.

In addition, effectors may play important roles in the pathogenicity of. During the infection, the plant pathogen secrets large amounts of effectors, which suppress host defense and induce the physiological changes in the host, to promote pathogen growth or spreading (Hogenhout et al, 2009). Among the 628 secreted proteins of, more than 18 predicted effectors have been observed to suppress hypersensitive response and 13 putative effectors are known to induce plant cell death in(Zhang et al, 2014; Fang et al, 2016). These findings suggest that the secreted proteins ofhave important roles in modulating plant defense mechanisms. However, the functions of theeffector genes during the pathogenic processes in rice remain unclarified.

Rice responses to U. virens infection

The formation of false smut balls requires specific interactions between rice and. By using transcriptional analyses, the molecular responses of rice in the compatible and incompatible interactions of rice-have been revealed. In the compatible interaction,prevents the production of pollen, ovary fertilization and flower-opening process to establish the infection (Chao et al, 2014; Fan et al, 2015; Song et al, 2016). Moreover, the infection ofdownregulates the expression levels of defense- related genes, such asand, to suppress the host defense (Fan et al, 2015). However, the infection ofactivates the expression of genes associated with grain filling, including the endosperm specific transcription factors, starch anabolism genes and seed storage protein genes, and fertilization, indicating thattakes advantage of rice nutrients supply systems by unknown mechanisms to form rice false smut balls (Fan et al, 2015; Song et al, 2016). Meanwhile, in the incompatible interaction, many pathogenesis related genes are induced to activate the rice resistance signaling pathways (Han et al, 2015). Taken together, these findings provide clues for that some factors fromcould potentially target the rice proteins or promoter regions of rice genes to regulate the expression levels of rice fertilization genes and/or pathogenesis related genes.

The utilization of highly resistant cultivars with resistance () genes/QTLs can provide an effective, economical and environmentally safe way to control plant diseases. In the 20 years, a number of reports have shown that some rice cultivars are highly resistant to rice false smut disease (Jin et al, 2005; Yang et al, 2008; Chen et al, 2009; Huang et al, 2010; Jiang et al, 2010; Lu et al, 2012; Lan et al, 2016). However, the results of the majority of these reports are based on artificial inoculation or natural infection data in the field lacking experimental replication and control of environmental conditions. It is well known that the environmental factors, such as temperature, humidity, nitrogen and sowing time, largely affect the severity of rice false smut disease (Wang et al, 2010; Liu et al, 2013; Wang W B et al, 2014a; Fu R T et al, 2015). Thus, it is difficult to come to any final conclusions regarding whether some of the rice cultivars are in fact highly resistant to rice false smut disease, or whether some of the rice cultivars havegenes for rice false smut disease. However, it has been confirmed that the resistance levels to rice false smut disease are obviously different among the rice cultivars. The resistance of rice to rice false smut disease may be provided bygenes and/or QTL. Some QTLshave been identified using recombinant inbred lines derived from a cross of IR28 and Daguandao, and the introgression lines from a cross between Teqing and Lemont (Li et al, 2011, 2014; Zhou et al, 2013).

Perspects

Currently, an increasing number of studies have been attempted to identify the resistance genes in rice and uncover the resistance mechanisms for rice false smut disease. In addition, the availability of thegenome sequence, along with efficient approach for targeted gene deletion and better artificial inoculation methods, have radically altered the methods by which the biology of rice false smut disease can be explored (Hu et al, 2014; Jia et al, 2015; Liang et al, 2018). Several important questions may be answered in the near future. For example, the infection processes ofin nature and whether there are anygenes in rice for rice false smut disease resistance breeding may be revealed. Other important research areas, such as howhijacks the nutrient supply in rice, as well as the functions of the effectors and pathogenic genes duringinfection processes are being currently investigated. Moreover, identification of the key genetic determinants ofconserved among different isolates which will provide the targets for new anti-fungal drugs in the future. In addition, further research will potentially know how rice false smut disease epidemics can be predicted by rapid and early detection methods. The results of the aforementioned research regardingwill offer references for the control of false smut disease in rice.

ACKNOWLEDGEMENTS

This study was funded by the Zhejiang Provincial Natural Science Foundation of China (Grant Nos. LQ19C140004 and LQ19C130007), the Chinese Academy of Agricultural Sciences under the ?Elite Youth? Program and the Agricultural Sciences and Technologies Innovation Program of China (CAAS-ASTIP-2016-CNRRI).

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1 July 2018;

29 October 2018

Kou Yanjun (kouyanjun@caas.cn)

Copyright ? 2019, China National Rice Research Institute. Hosting by Elsevier B V

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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http://dx.doi.org/10.1016/j.rsci.2018.10.007

(Managing Editor: Li Guan)