'Summary'	1001	
I.	'Live and let die – the basics of interaction'	1001	
II.	'A kettle of color – components of apoplastic plant defense'	1003	
III.	'To burst or not to burst – reactive oxygen in plant defense'	1004	
IV.	'How they learned to live with the bomb – disarming proteases'	1005	
V.	'Hide and seek – how to survive the apoplast'	1006	
VI.	'Effect or not – apoplastic virulence factors'	1007	
VII.	'Back to the future – challenges and opportunities'	1011	
 	'Acknowledgements'	1011	
 	References	1011	


Summary
Microbial plant pathogens have evolved a variety of strategies to enter plant hosts and cause disease. In particular, biotrophic pathogens, which parasitize living plant tissue, establish sophisticated interactions in which they modulate the plant's metabolism to their own good. The prime decision, whether or not a pathogen can accommodate itself in its host tissue, is made during the initial phase of infection. At this stage, the plant immune system recognizes conserved molecular patterns of the invading microbe, which initiate a set of basal immune responses. Induced plant defense proteins, toxic compounds and antimicrobial proteins encounter a broad arsenal of pathogen-derived virulence factors that aim to disarm host immunity. Crucial regulatory processes and protein–protein interactions take place in the apoplast, that is, intercellular spaces, plant cell walls and defined host–pathogen interfaces which are formed between the plant cytoplasm and the specialized infection structures of many biotrophic pathogens. This article aims to provide an insight into the most important principles and components of apoplastic plant immunity and its modulation by filamentous microbial pathogens.

https://nph.onlinelibrary.wiley.com/doi/pdf/10.1111/nph.12277

Apoplastic immunity and its suppression by filamentous plant pathogens.

Chitin is a major component of fungal cell wall and is synthesized by chitin synthases (Chs) Plant pathogenic fungi normally have multiple chitin synthase genes To determine their roles in development and pathogenesis, we functionally characterized all seven CHS genes in Magnaporthe oryzae Three of them, CHS1, CHS6, and CHS7, were found to be important for plant infection While the chs6 mutant was non-pathogenic, the chs1 and chs7 mutants were significantly reduced in virulence CHS1 plays a specific role in conidiogenesis, an essential step for natural infection cycle Most of chs1 conidia had no septum and spore tip mucilage The chs6 mutant was reduced in hyphal growth and conidiation It failed to penetrate and grow invasively in plant cells The two MMD-containing chitin synthase genes, CHS5 and CHS6, have a similar expression pattern Although deletion of CHS5 had no detectable phenotype, the chs5 chs6 double mutant had more severe defects than the chs6 mutant, indicating that they may have overlapping functions in maintaining polarized growth in vegetative and invasive hyphae Unlike the other CHS genes, CHS7 has a unique function in appressorium formation Although it was blocked in appressorium formation by germ tubes on artificial hydrophobic surfaces, the chs7 mutant still produced melanized appressoria by hyphal tips or on plant surfaces, indicating that chitin synthase genes have distinct impacts on appressorium formation by hyphal tip and germ tube The chs7 mutant also was defective in appressorium penetration and invasive growth Overall, our results indicate that individual CHS genes play diverse roles in hyphal growth, conidiogenesis, appressorium development, and pathogenesis in M oryzae, and provided potential new leads in the control of this devastating pathogen by targeting specific chitin synthases

https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1002526&type=printable

Different Chitin Synthase Genes Are Required for Various Developmental and Plant Infection Processes in the Rice Blast Fungus Magnaporthe oryzae

Genetic control of infection-related development in Magnaporthe oryzae.

Because of great challenges and workload in deleting genes on a large scale, the functions of most genes in pathogenic fungi are still unclear. In this study, we developed a high-throughput gene knockout system using a novel yeast-Escherichia-Agrobacterium shuttle vector, pKO1B, in the rice blast fungus Magnaporthe oryzae. Using this method, we deleted 104 fungal-specific Zn(2)Cys(6) transcription factor (TF) genes in M. oryzae. We then analyzed the phenotypes of these mutants with regard to growth, asexual and infection-related development, pathogenesis, and 9 abiotic stresses. The resulting data provide new insights into how this rice pathogen of global significance regulates important traits in the infection cycle through Zn(2)Cys(6)TF genes. A large variation in biological functions of Zn(2)Cys(6)TF genes was observed under the conditions tested. Sixty-one of 104 Zn(2)Cys(6) TF genes were found to be required for fungal development. In-depth analysis of TF genes revealed that TF genes involved in pathogenicity frequently tend to function in multiple development stages, and disclosed many highly conserved but unidentified functional TF genes of importance in the fungal kingdom. We further found that the virulence-required TF genes GPF1 and CNF2 have similar regulation mechanisms in the gene expression involved in pathogenicity. These experimental validations clearly demonstrated the value of a high-throughput gene knockout system in understanding the biological functions of genes on a genome scale in fungi, and provided a solid foundation for elucidating the gene expression network that regulates the development and pathogenicity of M. oryzae.

/pdf/systematic-analysis-of-zn2cys6-transcription-factors-44ayjbau3g.pdf

Systematic Analysis of Zn2Cys6 Transcription Factors Required for Development and Pathogenicity by High-Throughput Gene Knockout in the Rice Blast Fungus

Genome dynamics of pathogenic organisms are driven by pathogen and host co-evolution, in which pathogen genomes are shaped to overcome stresses imposed by hosts with various genetic backgrounds through generation of a variety of isolates. This same principle applies to the rice blast pathogen Magnaporthe oryzae and the rice host; however, genetic variations among different isolates of M. oryzae remain largely unknown, particularly at genome and transcriptome levels. Here, we applied genomic and transcriptomic analytical tools to investigate M. oryzae isolate 98-06 that is the most aggressive in infection of susceptible rice cultivars. A unique 1.4 Mb of genomic sequences was found in isolate 98-06 in comparison to reference strain 70-15. Genome-wide expression profiling revealed the presence of two critical expression patterns of M. oryzae based on 64 known pathogenicity-related (PaR) genes. In addition, 134 candidate effectors with various segregation patterns were identified. Five tested proteins could suppress BAX-mediated programmed cell death in Nicotiana benthamiana leaves. Characterization of isolate-specific effector candidates Iug6 and Iug9 and PaR candidate Iug18 revealed that they have a role in fungal propagation and pathogenicity. Moreover, Iug6 and Iug9 are located exclusively in the biotrophic interfacial complex (BIC) and their overexpression leads to suppression of defense-related gene expression in rice, suggesting that they might participate in biotrophy by inhibiting the SA and ET pathways within the host. Thus, our studies identify novel effector and PaR proteins involved in pathogenicity of the highly aggressive M. oryzae field isolate 98-06, and reveal molecular and genomic dynamics in the evolution of M. oryzae and rice host interactions.

/pdf/global-genome-and-transcriptome-analyses-of-magnaporthe-46n65ryidi.pdf

Global Genome and Transcriptome Analyses of Magnaporthe oryzae Epidemic Isolate 98-06 Uncover Novel Effectors and Pathogenicity-Related Genes, Revealing Gene Gain and Lose Dynamics in Genome Evolution

Saccharomyces cerevisiae Yap1 protein is an AP1-like transcription factor involved in the regulation of the oxidative stress response. An ortholog of Yap1, MoAP1, was recently identified from the rice blast fungus Magnaporthe oryzae genome. We found that MoAP1 is highly expressed in conidia and during invasive hyphal growth. The Moap1 mutant was sensitive to H2O2, similar to S. cerevisiae yap1 mutants, and MoAP1 complemented Yap1 function in resistance to H2O2, albeit partially. The Moap1 mutant also exhibited various defects in aerial hyphal growth, mycelial branching, conidia formation, the production of extracellular peroxidases and laccases, and melanin pigmentation. Consequently, the Moap1 mutant was unable to infect the host plant. The MoAP1-eGFP fusion protein is localized inside the nucleus upon exposure to H2O2, suggesting that MoAP1 also functions as a redox sensor. Moreover, through RNA sequence analysis, many MoAP1-regulated genes were identified, including several novel ones that were also involved in pathogenicity. Disruption of respective MGG_01662 (MoAAT) and MGG_02531 (encoding hypothetical protein) genes did not result in any detectable changes in conidial germination and appressorium formation but reduced pathogenicity, whereas the mutant strains of MGG_01230 (MoSSADH) and MGG_15157 (MoACT) showed marketed reductions in aerial hyphal growth, mycelial branching, and loss of conidiation as well as pathogenicity, similar to the Moap1 mutant. Taken together, our studies identify MoAP1 as a positive transcription factor that regulates transcriptions of MGG_01662, MGG_02531, MGG_01230, and MGG_15157 that are important in the growth, development, and pathogenicity of M. oryzae.

/pdf/the-bzip-transcription-factor-moap1-mediates-the-oxidative-1knvqh85la.pdf

The bZIP transcription factor MoAP1 mediates the oxidative stress response and is critical for pathogenicity of the rice blast fungus Magnaporthe oryzae.

Yan Du

Papers

The bZIP transcription factor MoAP1 mediates the oxidative stress response and is critical for pathogenicity of the rice blast fungus Magnaporthe oryzae.

Threonine deaminase MoIlv1 is important for conidiogenesis and pathogenesis in the rice blast fungus Magnaporthe oryzae.