Leaf rust, caused by Puccinia triticina , is one of the most destructive fungal diseases in wheat production worldwide. The hypersensitive reaction (HR) is an important defence response against P. triticina infection. In this study, the physiological races 165 and 260 of P. triticina were combined with a line derived from the bread wheat cultivar Thatcher with the leaf rust resistance locus Lr26 to form compatible and incompatible combinations, respectively. Based on an RNA‐Seq database of the interaction systems, a new wheat cysteine‐rich receptor‐like kinase gene, TaCRK2 , is specifically induced and up‐regulated in the incompatible combination. We identified that TaCRK2 was regulated in a Ca ²⁺ ‐dependent manner. Knockdown of TaCRK2 by virus‐induced gene silencing and RNAi leads to a dramatic increase in HR area and the number of haustorial mother cells at the single infection site. In addition, urediniospores, a P. triticina‐ specific pathogenic marker in compatible combinations, were observed on leaf surfaces of silenced plants at approximately 15 days after inoculation in the incompatible combination. Moreover, transcription levels of TaPR1, TaPR2 , and TaPR5 were obviously reduced in TaCRK2 ‐silenced plants. TaCRK2 overexpression in Nicotiana benthamiana induced strong HR‐like cell death. Finally, transient expression of green fluorescent protein fused with TaCRK2 in N. benthamiana indicated that TaCRK2 localizes in the endoplasmic reticulum. Thus, TaCRK2 plays an important role in the resistance to P. triticina infection and has a positive regulation effect on the HR cell death process induced by P. triticina .

While the QuikChange site-directed mutagenesis method and its later modifications are extremely useful and simple, they suffer from several drawbacks. Here, we propose a new method, named LFEAP mutagenesis (Ligation of Fragment Ends After PCR) for creating various mutations in plasmid by leveraging three existing concepts: inverse PCR, single primer PCR, and sticky-end assembly. The first inverse PCR on the target plasmid yielded linearized DNA fragments with mutagenic ends, and a second single primer PCR resulted in complementary single-stranded DNA fragments with the addition of overhangs at the 5′ end of each strand. The resulting single strands were then annealed to produce double-stranded DNA with free 5′ single-stranded DNA tails. These products with compatible sticky ends were efficiently assembled into a circular, mutagenized plasmid. With this strategy, multiple simultaneous changes (up to 15) and mutations in large plasmids (up to 50 kb) were achieved with high efficiency and fidelity. LFEAP mutagenesis is a versatile method that offers significant advantages for introducing large and multiple changes in plasmid DNA.

Background: Identification of the motifs bound by a transcription factor (TF) is important to reveal the function of TF. Previously, we built a transcription factor centered yeast one hybrid (TF-Centered Y1H) that could identify the motifs bound by a target TF. However, that method was difficult to comprehensively identify all the motifs bound by a TF. Results: Here, we build an improved TF-Centered Y1H to comprehensively determine the motifs bound by a target TF. Recombination-mediated cloning in yeast was performed to construct a saturated prey library that contains 7 random base insertions. After TF-Centered Y1H screening, all the positive clones were pooled together to isolate pHIS2 vector. The insertion regions of pHIS2 were PCR amplified and the PCR product was subjected to high-throughput sequencing. The insertion sequences were then retrieved and analyzed using MEME program to identify the potential motifs bound by the TF. Using this technology, we studied the motifs bound by an ethylene-responsive factor (BpERF2) from birch. In total, 22 conserved motifs were identified, and most of them are novel cis-acting elements. Both the yeast one hybrid and electrophoretic mobility shift assay verified that the obtained motifs could be bound by BpERF2. In addition, chromatin immunoprecipitation (ChIP) study further suggested that the identified motifs can be bound by BpERF2 in cells of birch. These results together suggested that this technology is reliable and has biological significance. Conclusion: This method will have wide application in DNA-protein interaction studies.

RNA polymerase II (RNAPII) is one of the central enzymes in cell growth and organizational development. It is a large macromolecular complex consisting of twelve subunits. Relative to the clear definition of RNAPII structure and biological function, the molecular mechanism of how RNAPII is assembled is poorly understood, because of that the key assembly factors acting for the assembly of RNAPII remain elusive. In this study, we identified two factors, Gpn2 and Rba50, which directly participate in the assembly of RNAPII. Gpn2 and Rba50 were demonstrated to interact with Rpb12 and Rpb3, respectively. The interaction between Gpn2 and Rba50 was also demonstrated. When Gpn2 and Rba50 are functionally defective, the assembly of the Rpb3 sub-complex is disrupted, leading to defect in the assembly of RNAPII. Based on these results, we conclude that Gpn2 and Rba50 directly participate in the assembly of the Rpb3 sub-complex and subsequently the biogenesis of RNAPII.

Eukaryotic organisms activate conserved signalling networks to maintain genomic stability in response to DNA genotoxic stresses. However, the coordination of this response pathway in fungal pathogens remains largely unknown. In the present study, we investigated the mechanism by which the northern corn leaf blight pathogen Setosphaeria turcica controls maize infection and activates self‐protection pathways in response to DNA genotoxic insults. Appressorium‐mediated maize infection by S. turcica was blocked by the S‐phase checkpoint. This repression was dependent on the checkpoint central kinase Ataxia Telangiectasia and Rad3 related (ATR), as inhibition of ATR activity or knockdown of the ATR gene recovered appressorium formation in the presence of genotoxic reagents. ATR promoted melanin biosynthesis in S. turcica as a defence response to stress. The melanin biosynthesis genes StPKS and StLac2 were induced by the ATR‐mediated S‐phase checkpoint. The responses to DNA genotoxic stress were conserved in a wide range of phytopathogenic fungi, including Cochliobolus heterostrophus , Cochliobolus carbonum , Alternaria solani , and Alternaria kikuchiana , which are known causal agents for plant diseases. We propose that in response to genotoxic stress, phytopathogenic fungi including S. turcica activate an ATR‐dependent pathway to suppress appressorium‐mediated infection and induce melanin‐related self‐protection in addition to conserved responses in eukaryotes.

A surveillance mechanism, the S phase checkpoint, blocks progression into mitosis in response to DNA damage and replication stress. Segregation of damaged or incompletely replicated chromosomes results in genomic instability. In humans, the S phase checkpoint has been shown to constitute an anti-cancer barrier. Inhibition of mitotic cyclin dependent kinase (M-CDK) activity by Wee1 kinases is critical to block mitosis in some organisms. However, such mechanism is dispensable in the response to genotoxic stress in the model eukaryotic organism Saccharomyces cerevisiae. We show here that the Wee1 ortholog Swe1 does indeed inhibit M-CDK activity and chromosome segregation in response to genotoxic insults. Swe1 dispensability in budding yeast is the result of a redundant control of M-CDK activity by the checkpoint kinase Rad53. In addition, our results indicate that Swe1 is an effector of the checkpoint central kinase Mec1. When checkpoint control on M-CDK and on Pds1/securin stabilization are abrogated, cells undergo aberrant chromosome segregation. Genetic inheritance during cell proliferation requires chromosome duplication (replication) and segregation of the replicated chromosomes to the two daughter cells. In response to the presence of DNA damage, cells block chromosome segregation to avoid the inheritance of damaged, incompletely replicated chromosomes. Failure to do so results in loss of genomic integrity. Here we show that three different, redundant pathways are responsible for such control in budding yeast, a model eukaryotic organism. One of the pathways had been described before and blocks the separation of the replicated chromosomes. We show now that two additional pathways inhibit the essential pro-mitotic Cyclin Dependent Kinase (M-CDK) activity. One of them involves the conserved inhibition of M-CDK through tyrosine phosphorylation, which was puzzlingly dispensable in the response to challenged replication in budding yeast. We show that the reason for such dispensability is the existence of redundant control of M-CDK activity by Rad53. Rad53 is part of a surveillance mechanism termed the S phase checkpoint that detects and responds to replication insults. Such control mechanism has been proposed to constitute an anti-cancer barrier in human cells.