Identification of Nicotiana benthamiana Genes Involved in Pathogen-Associated Molecular Pattern–Triggered Immunity

In order to identify components of pathogen-associated molecular pattern–triggered immunity (PTI) pathways in Nicotiana benthamiana, we conducted a large-scale forward-genetics screen using virus-induced gene silencing and a cell-death-based assay for assessing PTI. The assay relied on four combinations of PTI-inducing nonpathogens and cell-death-causing challenger pathogens and was first validated in plants silenced for FLS2 or BAK1. Over 3,200 genes were screened and 14 genes were identified that, when silenced, compromised PTI as judged by the cell-death-based assay. Further analysis indicated that the 14 genes were not involved in a general cell death response. A subset of the genes was found to act downstream of FLS2-mediated PTI induction, and silencing of three genes compromised production of reactive oxygen species in leaves exposed to flg22. The 14 genes encode proteins with potential functions in defense and hormone signaling, protein stability and degradation, energy and secondary metabolism, and cell wall biosynthesis and provide a new resource to explore the molecular basis for the involvement of these processes in PTI.

Download Full-text

A cell wall-localized glycine-rich protein of dodder acts as pathogen-associated molecular pattern

Communicative & Integrative Biology ◽

10.1080/19420889.2021.1918369 ◽

2021 ◽

Vol 14 (1) ◽

pp. 111-114

Author(s):

Peter Slaby ◽

Max Körner ◽

Markus Albert

Keyword(s):

Cell Wall ◽

Molecular Pattern ◽

A Cell ◽

Pathogen Associated Molecular Pattern ◽

Glycine Rich Protein

Download Full-text

The Effect of Transcription Factor MYB14 on Defense Mechanisms in Vitis quinquangularis-Pingyi

International Journal of Molecular Sciences ◽

10.3390/ijms21030706 ◽

2020 ◽

Vol 21 (3) ◽

pp. 706 ◽

Cited By ~ 5

Author(s):

Yangyang Luo ◽

Qingyang Wang ◽

Ru Bai ◽

Ruixiang Li ◽

Lu Chen ◽

...

Keyword(s):

Transcription Factor ◽

Nicotiana Benthamiana ◽

Defense Mechanisms ◽

Qualitative Difference ◽

Defense Responses ◽

Molecular Pattern ◽

Effector Triggered Immunity ◽

Pathogen Associated Molecular Pattern ◽

Basal Immunity ◽

Stilbene Biosynthesis

In the current study, we identified a transcription factor, MYB14, from Chinese wild grape, Vitis quinquangularis-Pingyi (V. quinquangularis-PY), which could enhance the main stilbene contents and expression of stilbene biosynthesis genes (StSy/RS) by overexpression of VqMYB14. The promoter of VqMYB14 (pVqMYB14) was shown to be induced as part of both basal immunity (also called pathogen-associated molecular pattern (PAMP)-triggered immunity, PTI) and effector-triggered immunity (ETI), triggered by the elicitors flg22 and harpin, respectively. This was demonstrated by expression of pVqMYB14 in Nicotiana benthamiana and Vitis. We identified sequence differences, notably an 11 bp segment in pVqMYB14 that is important for the PTI/ETI, and particularly for the harpin-induced ETI response. In addition, we showed that activation of the MYB14 promoter correlates with differences in the expression of MYB14 and stilbene pattern induced by flg22 and harpin. An experimental model of upstream signaling in V. quinquangularis-PY is presented, where early defense responses triggered by flg22 and harpin partially overlap, but where the timing and levels differ. This translates into a qualitative difference with respect to patterns of stilbene accumulation.

Download Full-text

Cell Death of Nicotiana benthamiana Is Induced by Secreted Protein NIS1 of Colletotrichum orbiculare and Is Suppressed by a Homologue of CgDN3

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-12-11-0316 ◽

2012 ◽

Vol 25 (5) ◽

pp. 625-636 ◽

Cited By ~ 33

Author(s):

Kae Yoshino ◽

Hiroki Irieda ◽

Fumie Sugimoto ◽

Hirofumi Yoshioka ◽

Tetsuro Okuno ◽

...

Keyword(s):

Cell Death ◽

Nicotiana Benthamiana ◽

Protein Gene ◽

Fluorescent Protein ◽

Expression System ◽

Functional Screening ◽

Secreted Protein ◽

Virus Induced Gene Silencing ◽

Colletotrichum Orbiculare ◽

Green Fluorescent

Colletotrichum orbiculare, the causal agent of cucumber anthracnose, infects Nicotiana benthamiana. Functional screening of C. orbiculare cDNAs in a virus vector-based plant expression system identified a novel secreted protein gene, NIS1, whose product induces cell death in N. benthamiana. Putative homologues of NIS1 are present in selected members of fungi belonging to class Sordariomycetes, Dothideomycetes, or Orbiliomycetes. Green fluorescent protein–based expression studies suggested that NIS1 is preferentially expressed in biotrophic invasive hyphae. NIS1 lacking signal peptide did not induce NIS1-triggered cell death (NCD), suggesting apoplastic recognition of NIS1. NCD was prevented by virus-induced gene silencing of SGT1 and HSP90, indicating the dependency of NCD on SGT1 and HSP90. Deletion of NIS1 had little effect on the virulence of C. orbiculare against N. benthamiana, suggesting possible suppression of NCD by C. orbiculare at the postinvasive stage. The CgDN3 gene of C. gloeosporioides was previously identified as a secreted protein gene involved in suppression of hypersensitive-like response in Stylosanthes guianensis. Notably, we found that NCD was suppressed by the expression of a CgDN3 homologue of C. orbiculare. Our findings indicate that C. orbiculare expresses NIS1 at the postinvasive stage and suggest that NCD could be repressed via other effectors, including the CgDN3 homologue.

Download Full-text

Apoptosis-induced alkalinization by the Na+/H+ exchanger isoform 1 is mediated through phosphorylation of amino acids Ser726 and Ser729

AJP Cell Physiology ◽

10.1152/ajpcell.00574.2007 ◽

2008 ◽

Vol 295 (4) ◽

pp. C883-C896 ◽

Cited By ~ 24

Author(s):

Amy L. Grenier ◽

Khaled Abu-ihweij ◽

Ge Zhang ◽

Shannon Moore Ruppert ◽

Rebecca Boohaker ◽

...

Keyword(s):

Cell Death ◽

P38 Map Kinase ◽

Serine Phosphorylation ◽

Targeted Mutagenesis ◽

Mutant Form ◽

Cytosolic Ph ◽

Complex Process ◽

Cell Death Response ◽

Transient Rise ◽

A Cell

Apoptosis is a complex process essential for normal tissue development and cellular homeostasis. While biochemical events that occur late in the apoptotic process are better characterized, early physiological changes that initiate the progression of cell death remain poorly understood. Previously, we observed that lymphocytes, undergoing apoptosis in response to growth factor withdrawal, experienced a rapid and transient rise in cytosolic pH. We found that the protein responsible was the pH-regulating, plasma membrane protein Na+/H+ exchanger isoform 1 (NHE1), and that its activity was impeded by inhibition of the stress-activated kinase, p38 MAP kinase. In the current study, we examined how NHE1 is activated during apoptosis. We identified the phosphorylation sites on NHE1 that regulate its alkalinizing activity in response to a cell death stimulus. Performing targeted mutagenesis, we observed that substitution of Ser726 and Ser729 for alanines produced a mutant form of NHE1 that did not alkalinize in response to an apoptotic stimulus, and expression of which protected cells from serum withdrawal- induced death. In contrast, substitution of Ser726 and Ser729 for glutamic acids raised the basal pH and induced susceptibility to death. Analysis of serine phosphorylation showed that phosphorylation of NHE1 during apoptosis decreased upon mutation of Ser726 and Ser729. Our findings thus confirm a necessary function for NHE1 during apoptosis and reveal the critical regulatory sites that when phosphorylated mediate the alkalinizing activity of NHE1 in the early stages of a cell death response.

Download Full-text

TIR domains of plant immune receptors are NAD+-cleaving enzymes that promote cell death

Science ◽

10.1126/science.aax1771 ◽

2019 ◽

Vol 365 (6455) ◽

pp. 799-803 ◽

Cited By ~ 69

Author(s):

Li Wan ◽

Kow Essuman ◽

Ryan G. Anderson ◽

Yo Sasaki ◽

Freddy Monteiro ◽

...

Keyword(s):

Cell Death ◽

Enzymatic Activity ◽

Interleukin 1 ◽

Adenosine Diphosphate ◽

Immune Receptors ◽

Cell Death Response ◽

A Cell ◽

Self Association ◽

Activate Cell ◽

Tir Domains

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors activate cell death and confer disease resistance by unknown mechanisms. We demonstrate that plant Toll/interleukin-1 receptor (TIR) domains of NLRs are enzymes capable of degrading nicotinamide adenine dinucleotide in its oxidized form (NAD+). Both cell death induction and NAD+ cleavage activity of plant TIR domains require known self-association interfaces and a putative catalytic glutamic acid that is conserved in both bacterial TIR NAD+-cleaving enzymes (NADases) and the mammalian SARM1 (sterile alpha and TIR motif containing 1) NADase. We identify a variant of cyclic adenosine diphosphate ribose as a biomarker of TIR enzymatic activity. TIR enzymatic activity is induced by pathogen recognition and functions upstream of the genes enhanced disease susceptibility 1 (EDS1) and N requirement gene 1 (NRG1), which encode regulators required for TIR immune function. Thus, plant TIR-NLR receptors require NADase function to transduce recognition of pathogens into a cell death response.

Download Full-text

Functional characterisation of the Nep1-like protein effectors of the necrotrophic pathogen - Alternaria brassicae

10.1101/2021.07.04.450447 ◽

2021 ◽

Author(s):

Deepak Duhan ◽

Shivani Gajbhiye ◽

Rajdeep Jaswal ◽

Ravindra Pal Singh ◽

Tilak Raj Sharma ◽

...

Keyword(s):

Cell Death ◽

Genome Sequence ◽

Systematic Study ◽

Subcellular Location ◽

Ethylene Biosynthesis ◽

Alternaria Brassicae ◽

Necrotrophic Pathogen ◽

Molecular Pattern ◽

Functional Characterisation ◽

Pathogen Associated Molecular Pattern

Alternaria brassicae is an important necrotrophic pathogen that infects the Brassicaceae family. A. brassicae, like other necrotrophs also secretes various proteinaceous effectors and metabolites that cause cell death to establish itself in the host. However, there has been no systematic study of A. brassicae effectors and their roles in pathogenesis. The availability of the genome sequence of A. brassicae has enabled the search for effectors and their functional characterisation. Nep1-like proteins are a superfamily of proteins that induce necrosis and ethylene biosynthesis. They have been reported from a variety of microbes including bacteria, fungi, and oomycetes. In this study, we identified two NLPs from A. brassicae viz. AbrNLP1 and AbrNLP2 and functionally characterised them. Although both AbrNLPs were found to be secretory in nature, they localised differentially inside the plant. AbrNLP2 was found to induce necrosis in both host and nonhost species, while AbrNLP1 could not induce necrosis in both species. Additionally, AbrNLP2 was shown to induce pathogen-associated molecular pattern (PAMP)-triggered immunity in both host and nonhost species. Overall, our study indicates that AbrNLPs are functionally and spatially (subcellular location) distinct and may play different but important roles during the pathogenesis of A. brassicae.

Download Full-text

Comparative transcriptomic analysis of candidate effectors to explore the infection and survival strategy of Bursaphelenchus xylophilus during different interaction stages with pine trees

BMC Plant Biology ◽

10.1186/s12870-021-02993-9 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Long-Jiao Hu ◽

Xiao-Qin Wu ◽

Xiao-Lei Ding ◽

Jian-Ren Ye

Keyword(s):

Nicotiana Benthamiana ◽

Cell Walls ◽

Bursaphelenchus Xylophilus ◽

Plant Signaling ◽

Host Defenses ◽

Molecular Pattern ◽

Pine Trees ◽

Pathogen Associated Molecular Pattern ◽

Comparative Transcriptomic Analysis ◽

Host Tissues

Abstract Background The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a devastating pathogen of many Pinus species in China. The aim of this study was to understand the interactive molecular mechanism of PWN and its host by comparing differentially expressed genes and candidate effectors from three transcriptomes of B. xylophilus at different infection stages. Results In total, 62, 69 and 46 candidate effectors were identified in three transcriptomes (2.5 h postinfection, 6, 12 and 24 h postinoculation and 6 and 15 d postinfection, respectively). In addition to uncharacterized pioneers, other candidate effectors were involved in the degradation of host tissues, suppression of host defenses, targeting plant signaling pathways, feeding and detoxification, which helped B. xylophilus survive successfully in the host. Seven candidate effectors were identified in both our study and the B. xylophilus transcriptome at 2.5 h postinfection, and one candidate effector was identified in all three transcriptomes. These common candidate effectors were upregulated at infection stages, and one of them suppressed pathogen-associated molecular pattern (PAMP) PsXEG1-triggered cell death in Nicotiana benthamiana. Conclusions The results indicated that B. xylophilus secreted various candidate effectors, and some of them continued to function throughout all infection stages. These various candidate effectors were important to B. xylophilus infection and survival, and they functioned in different ways (such as breaking down host cell walls, suppressing host defenses, promoting feeding efficiency, promoting detoxification and playing virulence functions). The present results provide valuable resources for in-depth research on the pathogenesis of B. xylophilus from the perspective of effectors.

Download Full-text