Limits...
Comparative genomic and transcriptional analyses of the carbohydrate-active enzymes and secretomes of phytopathogenic fungi reveal their significant roles during infection and development.

Lyu X, Shen C, Fu Y, Xie J, Jiang D, Li G, Cheng J - Sci Rep (2015)

Bottom Line: Our comparative genomic analysis showed that the numbers of plant cell wall (PCW)- and fungal cell wall (FCW)-degradation-associated carbohydrate-active enzymes (CAZymes) in necrotrophic and hemibiotrophic fungi are significantly larger than that in most biotrophic fungi.Furthermore, our results showed that the FCW reorganization or modification was also related to the fungal development.Among them, a small, cysteine-rich protein SsCVNH was experimentally confirmed to be essential for the virulence and sclerotial development, indicating that the small secreted proteins might also play crucial roles as potential effectors in host-non-specific necrotrophic fungi.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, Hubei Province, China.

ABSTRACT
Our comparative genomic analysis showed that the numbers of plant cell wall (PCW)- and fungal cell wall (FCW)-degradation-associated carbohydrate-active enzymes (CAZymes) in necrotrophic and hemibiotrophic fungi are significantly larger than that in most biotrophic fungi. However, our transcriptional analyses of CAZyme-encoding genes in Melampsora larici-populina, Puccinia graminis and Sclerotinia sclerotiorum showed that many genes encoding PCW- and FCW-degradation-associated CAZymes were significantly up-regulated during the infection of both necrotrophic fungi and biotrophic fungi, indicating an existence of a universal mechanism underlying PCW degradation and FCW reorganization or modification, which are both intimately involved in necrotrophic and biotrophic fungal infection. Furthermore, our results showed that the FCW reorganization or modification was also related to the fungal development. Additionally, our transcriptional analysis of the secretome of S. sclerotiorum showed that many secreted protein-encoding genes were dramatically induced during infection. Among them, a small, cysteine-rich protein SsCVNH was experimentally confirmed to be essential for the virulence and sclerotial development, indicating that the small secreted proteins might also play crucial roles as potential effectors in host-non-specific necrotrophic fungi.

No MeSH data available.


Related in: MedlinePlus

The phenotypes of SsCVNH-silenced transformants.(a) Colony morphology in SsCVNH-silenced transformants. Colonies were grown on PDA for 10 days at 20 °C. (b) SsCVNH-silenced transformants showing significantly reduced virulence on detached rapeseed (Brassica napus) leaves. Virulence was evaluated based on the lesion diameter at 20 °C for 48 h. (c) Comparison of the hyphal tips of SsCVNH-silenced transformants and the wild-type strain. Scale bar = 100 μm. (d) Expression of SsCVNH in silenced transformants was determined by qRT-PCR. The expression levels of β-tubulin were used to normalize SsCVNH expression. The expression level of SsCVNH in the wild-type strain was set at 1.0. (e) Comparison of the lesion diameters of the silenced transformants and the wild-type strain. (f) Comparison of the growth rates of the silenced transformants and the wild-type strain. In all experiments, three independent replications were performed. The values are presented as the means ± s.d. Different letters in the graph indicate significant differences while same letters indicate no significant differences, P = 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4632110&req=5

f8: The phenotypes of SsCVNH-silenced transformants.(a) Colony morphology in SsCVNH-silenced transformants. Colonies were grown on PDA for 10 days at 20 °C. (b) SsCVNH-silenced transformants showing significantly reduced virulence on detached rapeseed (Brassica napus) leaves. Virulence was evaluated based on the lesion diameter at 20 °C for 48 h. (c) Comparison of the hyphal tips of SsCVNH-silenced transformants and the wild-type strain. Scale bar = 100 μm. (d) Expression of SsCVNH in silenced transformants was determined by qRT-PCR. The expression levels of β-tubulin were used to normalize SsCVNH expression. The expression level of SsCVNH in the wild-type strain was set at 1.0. (e) Comparison of the lesion diameters of the silenced transformants and the wild-type strain. (f) Comparison of the growth rates of the silenced transformants and the wild-type strain. In all experiments, three independent replications were performed. The values are presented as the means ± s.d. Different letters in the graph indicate significant differences while same letters indicate no significant differences, P = 0.05.

Mentions: Due to the presence of the multi-nucleated cells in S. sclerotiorum, RNAi technology was used to characterize the biological functions of SsCVNH. QRT-PCR was used to examine the transcript accumulation in SsCVNH-silenced transformants. The results showed that the sclerotial development of the transformants (SsCVNH-89, SsCVNH-84 and SsCVNH-40) with dramatically reduced SsCVNH expression was completely blocked on PDA medium at 20 °C. However, the wild-type strain and the transformant SsCVNH-46 with slightly reduced SsCVNH expression showed normal sclerotial development (Fig. 8a,d). The growth rate of the silenced transformants was also significantly reduced, although there was no obvious difference between the morphology of the hyphal tips of the silenced transformants and the wild-type strain (Fig. 8c,f). The virulence of the SsCVNH-silenced transformants was significantly reduced, and only small lesions developed on the detached leaves of Brassica napus at 2 dpi. Furthermore, the decrease in virulence was positively correlated with the silencing efficiency of different transformants (Fig. 8b,e). Similar result was observed when the SsCVNH-silenced transformants were inoculated on detached tomato leaves (Figure S8), indicating that the virulence reduction was not host species-specific. These results indicated that SsCVNH played crucial roles in the virulence, sclerotial development and growth rate of S. sclerotiorum. In conclusion, our results indicated that small secreted proteins in typically necrotrophic fungi might function as potential effectors similar to those in hemibiotrophs and biotrophs.


Comparative genomic and transcriptional analyses of the carbohydrate-active enzymes and secretomes of phytopathogenic fungi reveal their significant roles during infection and development.

Lyu X, Shen C, Fu Y, Xie J, Jiang D, Li G, Cheng J - Sci Rep (2015)

The phenotypes of SsCVNH-silenced transformants.(a) Colony morphology in SsCVNH-silenced transformants. Colonies were grown on PDA for 10 days at 20 °C. (b) SsCVNH-silenced transformants showing significantly reduced virulence on detached rapeseed (Brassica napus) leaves. Virulence was evaluated based on the lesion diameter at 20 °C for 48 h. (c) Comparison of the hyphal tips of SsCVNH-silenced transformants and the wild-type strain. Scale bar = 100 μm. (d) Expression of SsCVNH in silenced transformants was determined by qRT-PCR. The expression levels of β-tubulin were used to normalize SsCVNH expression. The expression level of SsCVNH in the wild-type strain was set at 1.0. (e) Comparison of the lesion diameters of the silenced transformants and the wild-type strain. (f) Comparison of the growth rates of the silenced transformants and the wild-type strain. In all experiments, three independent replications were performed. The values are presented as the means ± s.d. Different letters in the graph indicate significant differences while same letters indicate no significant differences, P = 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4632110&req=5

f8: The phenotypes of SsCVNH-silenced transformants.(a) Colony morphology in SsCVNH-silenced transformants. Colonies were grown on PDA for 10 days at 20 °C. (b) SsCVNH-silenced transformants showing significantly reduced virulence on detached rapeseed (Brassica napus) leaves. Virulence was evaluated based on the lesion diameter at 20 °C for 48 h. (c) Comparison of the hyphal tips of SsCVNH-silenced transformants and the wild-type strain. Scale bar = 100 μm. (d) Expression of SsCVNH in silenced transformants was determined by qRT-PCR. The expression levels of β-tubulin were used to normalize SsCVNH expression. The expression level of SsCVNH in the wild-type strain was set at 1.0. (e) Comparison of the lesion diameters of the silenced transformants and the wild-type strain. (f) Comparison of the growth rates of the silenced transformants and the wild-type strain. In all experiments, three independent replications were performed. The values are presented as the means ± s.d. Different letters in the graph indicate significant differences while same letters indicate no significant differences, P = 0.05.
Mentions: Due to the presence of the multi-nucleated cells in S. sclerotiorum, RNAi technology was used to characterize the biological functions of SsCVNH. QRT-PCR was used to examine the transcript accumulation in SsCVNH-silenced transformants. The results showed that the sclerotial development of the transformants (SsCVNH-89, SsCVNH-84 and SsCVNH-40) with dramatically reduced SsCVNH expression was completely blocked on PDA medium at 20 °C. However, the wild-type strain and the transformant SsCVNH-46 with slightly reduced SsCVNH expression showed normal sclerotial development (Fig. 8a,d). The growth rate of the silenced transformants was also significantly reduced, although there was no obvious difference between the morphology of the hyphal tips of the silenced transformants and the wild-type strain (Fig. 8c,f). The virulence of the SsCVNH-silenced transformants was significantly reduced, and only small lesions developed on the detached leaves of Brassica napus at 2 dpi. Furthermore, the decrease in virulence was positively correlated with the silencing efficiency of different transformants (Fig. 8b,e). Similar result was observed when the SsCVNH-silenced transformants were inoculated on detached tomato leaves (Figure S8), indicating that the virulence reduction was not host species-specific. These results indicated that SsCVNH played crucial roles in the virulence, sclerotial development and growth rate of S. sclerotiorum. In conclusion, our results indicated that small secreted proteins in typically necrotrophic fungi might function as potential effectors similar to those in hemibiotrophs and biotrophs.

Bottom Line: Our comparative genomic analysis showed that the numbers of plant cell wall (PCW)- and fungal cell wall (FCW)-degradation-associated carbohydrate-active enzymes (CAZymes) in necrotrophic and hemibiotrophic fungi are significantly larger than that in most biotrophic fungi.Furthermore, our results showed that the FCW reorganization or modification was also related to the fungal development.Among them, a small, cysteine-rich protein SsCVNH was experimentally confirmed to be essential for the virulence and sclerotial development, indicating that the small secreted proteins might also play crucial roles as potential effectors in host-non-specific necrotrophic fungi.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, Hubei Province, China.

ABSTRACT
Our comparative genomic analysis showed that the numbers of plant cell wall (PCW)- and fungal cell wall (FCW)-degradation-associated carbohydrate-active enzymes (CAZymes) in necrotrophic and hemibiotrophic fungi are significantly larger than that in most biotrophic fungi. However, our transcriptional analyses of CAZyme-encoding genes in Melampsora larici-populina, Puccinia graminis and Sclerotinia sclerotiorum showed that many genes encoding PCW- and FCW-degradation-associated CAZymes were significantly up-regulated during the infection of both necrotrophic fungi and biotrophic fungi, indicating an existence of a universal mechanism underlying PCW degradation and FCW reorganization or modification, which are both intimately involved in necrotrophic and biotrophic fungal infection. Furthermore, our results showed that the FCW reorganization or modification was also related to the fungal development. Additionally, our transcriptional analysis of the secretome of S. sclerotiorum showed that many secreted protein-encoding genes were dramatically induced during infection. Among them, a small, cysteine-rich protein SsCVNH was experimentally confirmed to be essential for the virulence and sclerotial development, indicating that the small secreted proteins might also play crucial roles as potential effectors in host-non-specific necrotrophic fungi.

No MeSH data available.


Related in: MedlinePlus