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Background and aims: Compared with tyrosine kinase inhibitor (TKI) monotherapy, TKI combined with PD1 can improve the therapeutic effect of liver cancer and has been widely used in clinical practice. However, there is a lack of effective biomarkers to identify patients who would benefit more from this combination therapy. Therefore, this study aimed to evaluate whether baseline lymphocyte counts can identify patients with liver cancer who would benefit from targeted immune combination therapy. Methods: Data from patients with hepatocellular carcinoma (HCC) who received TKIs or TKIs in combination with PD1 between June 2018 and June 2020 were retrospectively collected. The patients were divided into high and low groups based on the median absolute count of peripheral lymphocytes before systemic therapy and differences in overall survival (OS) and progression-free survival (PFS) between TKI and TKI+PD1 were compared between the two groups. Results: In total, 72 patients were included in this study, with a median follow-up of 1.5 years. Both PFS and OS in the TKI+PD1 group showed a good prognostic trend (p = 0.058 and p = 0.077, respectively). Subgroup analyses based on peripheral blood lymphocyte counts showed that the combination regimen had a significant PFS and OS advantage only in patients with high peripheral blood lymphocyte counts (p = 0.036 and p = 0.031, respectively), but not in patients with low absolute peripheral blood lymphocyte counts (p = 0.819 and p = 0.913, respectively). Conclusions: Peripheral blood lymphocyte count is a simple and effective biomarker that can be used to identify patients with liver cancer who will benefit more from TKI+PD-1 combination therapy. [ABSTRACT FROM AUTHOR]

Indole-3-acetic acid (IAA) belongs to the family of auxin indole derivatives. IAA regulates almost all aspects of plant growth and development, and is one of the most important plant hormones. In microorganisms too, IAA plays an important role in growth, development, and even plant interaction. Therefore, mechanism studies on the biosynthesis and functions of IAA in microorganisms can promote the production and utilization of IAA in agriculture. This mini-review mainly summarizes the biosynthesis pathways that have been reported in microorganisms, including the indole-3-acetamide pathway, indole-3-pyruvate pathway, tryptamine pathway, indole-3-acetonitrile pathway, tryptophan side chain oxidase pathway, and non-tryptophan dependent pathway. Some pathways interact with each other through common key genes to constitute a network of IAA biosynthesis. In addition, functional studies of IAA in microorganisms, divided into three categories, have also been summarized: the effects on microorganisms, the virulence on plants, and the beneficial impacts on plants. [ABSTRACT FROM AUTHOR]

The basal transcription factor II H (TFIIH) is a multicomponent complex. In the present study, we characterized a TFIIH subunit Tfb5 by analysing loss‐ and gain‐of‐function mutants to gain a better understanding of the molecular mechanisms underlying stress resistance and pathogenicity in the citrus fungal pathogen Alternaria alternata. Tfb5 deficiency mutants (ΔAatfb5) decreased sporulation and pigmentation, and were impaired in the maintenance of colony surface hydrophobicity and cell wall integrity. ΔAatfb5 increased sensitivity to ultraviolet light, DNA‐damaging agents, and oxidants. The expression of Aatfb5 was up‐regulated in the wild type upon infection in citrus leaves, implicating the requirement of Aatfb5 in fungal pathogenesis. Biochemical and virulence assays revealed that ΔAatfb5 was defective in toxin production and cellwall‐degrading enzymes, and failed to induce necrotic lesions on detached citrus leaves. Aatfb5 fused with green fluorescent protein (GFP) was localized in the cytoplasm and nucleus and physically interacted with another subunit, Tfb2, based on yeast two‐hybrid and co‐immunoprecipitation analyses. Transcriptome and Antibiotics & Secondary Metabolite Analysis Shell (antiSMASH) analyses revealed the positive and negative roles of Aatfb5 in the production of various secondary metabolites and in the regulation of many metabolic and biosynthetic processes in A. alternata. Aatfb5 may play a negative role in oxidative phosphorylation and a positive role in peroxisome biosynthesis. Two cutinase‐coding genes (AaCut2 and AaCut15) required for full virulence were down‐regulated in ΔAatfb5. Overall, this study expands our understanding of how A. alternata uses the basal transcription factor to deal with stress and achieve successful infection in the plant host. [ABSTRACT FROM AUTHOR]

Horizontal gene transfer (HGT) has been identified as an important source of genomic innovation in fungi. However, how HGT drove the evolution of Alternaria alternata , a necrotrophic fungus which can be ubiquitously isolated from soil and various plants and decaying plant materials is largely known. In this study, we identified 12 protein-encoding genes that are likely acquired from lineages outside Pezizomycotina. Phylogenetic trees and approximately unbiased comparative topology tests strongly supported the evolutionary origin of these genes. According to their predicted functions, these HGT candidates are involved in nitrogen and carbohydrate metabolism. Especially, five genes of them were likely transferred as a physically linked cluster from Tremellales (Basidiomycota). Functionally knocking out the five-gene cluster in an A. alternata isolate causing citrus brown spot resulted in an 80% decrease in asexual spore production in the deletion mutant. We further knocked out each of these five genes in this cluster and the resultant single-gene deletion mutants exhibited a various degree of reduction in spore production. Except for conidiation, functions of these genes associated with vegetative growth, stress tolerance, and virulence are very limited. Our results provide new evidence that HGT has played important roles over the course of the evolution of filamentous fungi. [ABSTRACT FROM AUTHOR]

Summary: The tangerine pathotype of the ascomycete fungus Alternaria alternata is the causal agent of citrus brown spot, which can result in significant losses of both yield and marketability for tangerines worldwide. A conditionally dispensable chromosome (CDC), which harbours the host‐selective ACT toxin gene cluster, is required for tangerine pathogenicity of A. alternata. To understand the genetic makeup and evolution of the tangerine pathotype CDC, we isolated and sequenced the CDCs of the A. alternata Z7 strain and analysed the function and evolution of their genes. The A. alternata Z7 strain has two CDCs (~1.1 and ~0.8 Mb, respectively), and the longer Z7 CDC contains all but one contig of the shorter one. Z7 CDCs contain 254 predicted protein‐coding genes, which are enriched in functional categories associated with 'metabolic process' (55 genes, P = 0.037). Relatively few of the CDC genes can be classified as carbohydrate‐active enzymes (CAZymes) (4) and transporters (19) and none as kinases. Evolutionary analysis of the 254 CDC proteins showed that their evolutionary conservation tends to be restricted within the genus Alternaria and that the CDC genes evolve faster than genes in the essential chromosomes, likely due to fewer selective constraints. Interestingly, phylogenetic analysis suggested that four of the 25 genes responsible for the ACT toxin production were likely transferred from Colletotrichum (Sordariomycetes). Functional experiments showed that two of them are essential for the virulence of the tangerine pathotype of A. alternata. These results provide new insights into the function and evolution of CDC genes in Alternaria. [ABSTRACT FROM AUTHOR]

The COP9 signalosome (CSN) is a highly conserved protein complex involved in the ubiquitin-proteasome system. Its metalloisopeptidase activity resides in subunit 5 (CSN5). Functions of csn5 in phytopathogenic fungi are poorly understood. Here, we knocked out the csn5 ortholog (Aacsn5) in the tangerine pathotype of Alternaria alternata. The ΔAacsn5 mutant showed a moderately reduced growth rate compared to the wildtype strain and was unable to produce conidia. The growth of ΔAacsn5 mutant was not affected in response to oxidative and osmotic stresses. Virulence assays revealed that ΔAacsn5 induced no or significantly reduced necrotic lesions on detached citrus leaves. The defects in hyphal growth, conidial sporulation, and pathogenicity of ΔAacsn5 were restored by genetic complementation of the mutant with wildtype Aacsn5. To explore the molecular mechanisms of conidiation and pathogenesis underlying Aacsn5 regulation, we systematically examined the transcriptomes of both ΔAacsn5 and the wildtype. Generally, 881 genes were overexpressed and 777 were underexpressed in the ΔAacsn5 mutant during conidiation while 694 overexpressed and 993 underexpressed during infection. During asexual development, genes related to the transport processes and nitrogen metabolism were significantly downregulated; the expression of csn1-4 and csn7 in ΔAacsn5 was significantly elevated; secondary metabolism gene clusters were broadly affected; especially, the transcript level of the whole of cluster 28 and 30 was strongly induced. During infection, the expression of the host-specific ACT toxin gene cluster which controls the biosynthesis of the citrus specific toxin was significantly repressed; many other SM clusters with unknown products were also regulated; 86 out of 373 carbohydrate-active enzymes responsible for breaking down the plant dead tissues showed uniquely decreased expression. Taken together, our results expand our understanding of the roles of csn5 on conidiation and pathogenicity in plant pathogenic fungi and provide a foundation for future investigations. [ABSTRACT FROM AUTHOR]

Subtilisin-like serine protease secreted by pathogenic fungi can facilitate the infection and acquisition of nutrients. Functions of subtilisin-like serine proteases in the phytopathogenic fungus Alternaria alternata remains unknown. In the current study, 15 subtilisin-like serine proteases were individually deleted in the citrus fungal pathogen A. alternata. Only one, designated AaPrb1, was found to be required for A. alternata pathogenesis. The AaPrb1 deficiency strain (Δ Aaprb1) reduced growth, conidiation, the formation of aerial hyphae, protease production, and virulence on citrus leaves. However, biochemical analyses and bioassays revealed that Δ Aaprb1 plays no role in the production of ACT toxin. Through Y2H assays, Aaprb1 was found to interact with Aapep4, a vacuole-localized proteinase A in A. alternata. Furthermore, silencing AaPep4 in A. alternata resulted in phenotypes similar with those of Δ Aaprb1. Expression of AaPrb1 was found to be regulated by AaPep4. TEM showed that AaPrb1and AaPep4 were involved in the suppression of the degradation of autophagosomes. Deletion of the autophagy gene AaAtg8 in A. alternata decreased conidiation, the formation of aerial hyphae and pathogenicity similar to Δ Aaprb1 , implying that some phenotypes of Δ Aaprb1 were due to the impairment of autophagy. Overall, this study expands our understanding of how A. alternata utilizes the subtilisin-like serine protease to achieve successful infection in the plant host. [ABSTRACT FROM AUTHOR]

This study determined the function of thioredoxin and glutaredoxin systems in the phytopathogenic fungus Alternaria alternata via analyzing mutants obtained from the targeted deletion of genes encoding thioredoxin peroxidase ( Tsa1 ), thioredoxin reductase ( Trr1 ), and glutathione reductase ( Glr1 ). Trr1 and Glr1 , but not Tsa1 , are required for growth and conidiation. The reduced growth and conidiation seen in the Trr1 or Glr1 deletion mutant can be restored by glutathione. Deletion mutants showing growth inhibition by oxidants are defective for H 2 O 2 detoxification and induce smaller lesions on citrus leaves. Trr1 and Glr1 , but not Tsa1 , also contribute to NaCl resistance. Glr1 is required for sorbitol resistance and is responsible for resistance to mancozeb and boscalid but not chlorothalonil fungicides, a novel phenotype that has not been reported in fungi. Trr1 is required for resistance to boscalid and chlorothalonil fungicides but confers susceptibility to mancozeb. The Tsa1 deletion mutant displays wild-type sensitivity to the tested fungicides. The expression of Tsa1 and Trr1 is regulated by the oxidative stress responsive regulators Yap1, Hog1, and Skn7. The expression of Tsa1 , but not Trr1 , is also regulated indirectly by the NADPH oxidase. The results indicate that the capability to resist oxidative stress is required for virulence of A. alternata IMPORTANCE The thioredoxin and glutaredoxin systems are important thiol antioxidant systems in cells, and knowledge of these two systems in the plant-pathogenic fungus A. alternata is useful for finding new strategies to reduce the virulence of this pathogen. In this study, we demonstrated that thiol antioxidant system-related genes ( Tsa1 , Trr1 , and Glr1 ) are required for H 2 O 2 detoxification and virulence in A. alternata Moreover, deletion of Trr1 results in hypersensitivity to the fungicides chlorothalonil and boscalid, and Glr1 deletion mutants are highly sensitive to mancozeb, which is the fungicide mostly used in citrus fields. Therefore, our findings demonstrate that the ability to detoxify reactive oxygen species (ROS) plays a critical role in pathogenesis on citrus and provide novel insights into the physiological functions of thiol-containing systems in fungicide sensitivity for A. alternata ., (Copyright © 2018 American Society for Microbiology.)