Clathrin Light Chains negatively regulate plant immunity by hijacking the autophagy pathway.

The crosstalk between clathrin-mediated endocytosis (CME) and autophagy pathway has been reported in mammals. However, the interconnection of CME with autophagy has not been established in plants. In this report, we showed that Arabidopsis CLATHRIN LIGHT CHAIN (CLC) subunit 2 and 3 double mutant, clc2-1 clc3-1, phenocopied the Arabidopsis AUTOPHAGY-RELATED GENE (ATG) mutants both in auto-immunity and nutrient sensitivity. Accordingly, the autophagy pathway was significantly compromised in the clc2-1 clc3-1 mutant. Interestingly, we demonstrated with multiple assays that CLC2 directly interacted with ATG8h/ATG8i in a domain-specific manner. As expected, both GFP-ATG8h/GFP-ATG8i and CLC2-GFP were subjected to autophagic degradation and the degradation of GFP-ATG8h was significantly reduced in the clc2-1 clc3-1 mutant. Notably, simultaneously knocking out ATG8h and ATG8i by the CRISPR/CAS9 resulted in an enhanced resistance against Golovinomyces cichoracearum, supporting the functional relevance of the CLC2-ATG8h/8i interactions. In conclusion, our results uncovered a link between the function of CLCs and the autophagy pathway in Arabidopsis.

[GmAGB1 plays a positive regulatory role in soybean defense responses].

Heterotrimeric GTP-binding protein (G-proteins) complex, which consists of Gα, Gß and Gγ subunits, plays critical roles in defense signaling. Arabidopsis genome contains only a single Gß-encoding gene, AGB1. Loss function of AGB1 in Arabidopsis results in enhanced susceptibility to a wide range of pathogens. However, the function of soybean AGB1 in immunity has not been previously interrogated. Bioinformatic analysis indicated that there are four GmAGB1 homologous genes in soybean genome, sharing homology of 86%-97%. To overcome the functional redundancy of these GmAGB1 homologs, virus-induced gene silencing (VIGS) mediated by the bean pod mottle virus (BPMV) was used to silence these four genes simultaneously. As expected, these four GmAGB1 homologous genes were indeed silenced by a single BPMV-VIGS vector carrying a conserved fragments among these four genes. A dwarfed phenotype was observed in GmAGB1s-silenced soybean plants, suggesting that GmAGB1s play a crucial role in growth and development. Disease resistance analysis indicated that silencing GmAGB1s significantly compromised the resistance of soybean plants against Xanthomonas campestris pv. glycinea (Xag). This reduced resistance was correlated with the decreased accumulation of pathogen-induced reactive oxygen species (ROS) and the reduced activation of GmMPK3 in response to flg22, a conserved N-terminal peptide of flagellin protein. These results indicate that GmAGB1 functions as a positive regulator in disease resistance and GmAGB1 is indispensable for the ROS production and GmMPK3 activation induced by pathogen infection. Yeast two hybrid assay showed that GmAGB1 interacted with GmAGG1, suggesting that an evolutionary conserved heterotrimeric G protein complex similarly functions in soybean.

Partially knocking out NtPDK1a/1b/1c/1d simultaneously in Nicotiana tabacum using CRISPR/CAS9 technology results in auxin-related developmental defects.

The eukaryotic AGC protein kinase subfamily (protein kinase A/ protein kinase G/ protein kinase C-family) is involved in regulating numerous biological processes across kingdoms, including growth and development, and apoptosis. PDK1(3-phosphoinositide-dependent protein kinase 1) is a conserved serine/threonine kinase in eukaryotes, which is both a member of AGC kinase and a major regulator of many other downstream AGC protein kinase family members. Although extensively investigated in model plant Arabidopsis, detailed reports for tobacco PDK1s have been limited. To better understand the functions of PDK1s in tobacco, CRISPR/CAS9 transgenic lines were generated in tetraploid N. tabacum, cv. Samsun (NN) with 5-7 of the 8 copies of 4 homologous PDK1 genes in tobacco genome (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out. Numerous developmental defects were observed in these NtPDK1a/1b/1c/1d CRISPR/CAS9 lines, including cotyledon fusion leaf shrinkage, uneven distribution of leaf veins, convex veins, root growth retardation, and reduced fertility, all of which reminiscence of impaired polar auxin transport. The severity of these defects was correlated with the number of knocked out alleles of NtPDK1a/1b/1c/1d. Consistent with the observation in Arabidopsis, it was found that the polar auxin transport, and not auxin biosynthesis, was significantly compromised in these knockout lines compared with the wild type tobacco plants. The fact that no homozygous plant with all 8 NtPDK1a/1b/1c/1d alleles being knocked out suggested that knocking out 8 alleles of NtPDK1a/1b/1c/1d could be lethal. In conclusion, our results indicated that NtPDK1s are versatile AGC kinases that participate in regulation of tobacco growth and development via modulating polar auxin transport. Our results also indicated that CRISPR/CAS9 technology is a powerful tool in resolving gene redundancy in polyploidy plants.

Knocking out NtSARD1a/1b/1c/1d by CRISPR/CAS9 technology reduces the biosynthesis of salicylic acid (SA) and compromises immunity in tetraploid Nicotiana tabacum.

Salicylic acid (SA) is a key phyto-hormone that is essential for plant immunity. SARD1 (SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1), a member of the CBP60 (CALMODULIN-BINDING PROTEIN60) gene family, is one of the major transcription factors regulating the expression of the genes in SA biosynthesis. SARD1 has been extensively studied in model plant Arabidopsis. However, the function of SARD1 homologues in SA biosynthesis and immune responses have rarely been investigated in other plant species. In this study, the CRISPR/CAS9 (Clustered Regularly Interspersed Short Palindromic Repeats/CAS9) technology was used in creating transgenic tobacco mutant lines with 6-8 alleles of four NtSARD1 homologous genes (NtSARD1a/1b/1c/1d) knocked out. No significant difference in morphological phenotype was observed between the transgenic knockout lines and the wild type tobacco plants, indicating that knocking out NtSARD1s does not affect the growth and development in tobacco. However, knocking out or partially knocking out of NtSARD1a/b/c/d resulted in a significantly reduced expression of NtICS1, the key gene in SA biosynthesis pathway, and thus the subsequently decreased SA/SAG accumulations in response to Pst DC3000 (Pseudomonas syrangae pv.tomato DC3000) infection, indicating a key role of NtSARD1 genes in SA biosynthesis in tobacco. As a consequence of reduced SA/SAG accumulation, the Pst DC3000-induced expression of NtPR genes as well as the resistance to Pst DC3000 were both significantly reduced in these knockout lines compared with the wild type tobacco plants. Interestingly, the reductions in the SA/SAG level, NtPR gene induction and Pst DC3000 resistance were positively correlated with the number of alleles being knocked out. Furthermore, LUC reporter gene driven by the promoter of NtICS1 containing two G(A/T)AATT(T/G) motifs could be activated by NtSARD1a, suggesting that NtSARD1a could bind to the core G(A/T)AATT(T/G) motifs and thus activate the expression of LUC reporter. Taken together, our results demonstrated that the NtSARD1 proteins play essential roles in SA biosynthesis and immune responses in tobacco. Our results also demonstrated that the CRISPR/CAS9 technology can overcome gene redundancy and is a powerful tool to study gene functions in polyploid plant species.

Silencing GmATG7 Leads to Accelerated Senescence and Enhanced Disease Resistance in Soybean.

Autophagy plays a critical role in nutrient recycling/re-utilizing under nutrient deprivation conditions. However, the role of autophagy in soybeans has not been intensively investigated. In this study, the Autophay-related gene 7 (ATG7) gene in soybeans (referred to as GmATG7) was silenced using a virus-induced gene silencing approach mediated by Bean pod mottle virus (BPMV). Our results showed that ATG8 proteins were highly accumulated in the dark-treated leaves of the GmATG7-silenced plants relative to the vector control leaves (BPMV-0), which is indicative of an impaired autophagy pathway. Consistent with the impaired autophagy, the dark-treated GmATG7-silenced leaves displayed an accelerated senescence phenotype, which was not seen on the dark-treated BPMV-0 leaves. In addition, the accumulation levels of both H2O2 and salicylic acid (SA) were significantly induced in the GmATG7-silenced plants compared with the BPMV-0 plants, indicating an activated immunity. Consistently, the GmATG7-silenced plants were more resistant against both Pseudomonas syringae pv. glycinea (Psg) and Soybean mosaic virus (SMV) compared with the BPMV-0 plants. However, the activated immunity in the GmATG7-silenced plant was not dependent upon the activation of MPK3/MPK6. Collectively, our results demonstrated that the function of GmATG7 is indispensable for autophagy in soybeans, and the activated immunity in the GmATG7-silenced plant is a result of impaired autophagy.

[Silencing GmATG10 results in activation of immune responses in soybean].

Autophagy is a highly conserved mechanism for material degradation and recycling in eukaryote cells, and plays important roles in growth, development, stress tolerance and immune responses. ATG10 plays a key role in autophagosome formation. To understand the function of ATG10 in soybean, two homologous GmATG10 genes, namely GmATG10a and GmATG10b, were silenced simultaneously by bean pod mottle virus (BPMV) induced gene silencing. The carbon starvation induced by dark treatment and Western blotting analysis of GmATG8 accumulation level indicated that concurrent silencing GmATG10a/10b resulted in the impairment of autophagy in soybean; disease resistance and kinase assays demonstrated that GmATG10a/10b participated in the immune responses by negatively regulating the activation of GmMPK3/6, indicating that GmATG10a/10b plays a negative regulatory role in immune response in soybean.

GmCBP60b Plays Both Positive and Negative Roles in Plant Immunity.

CBP60b (CALMODULIN-BINDING PROTEIN 60b) is a member of the CBP60 transcription factor family. In Arabidopsis, AtCBP60b not only regulates growth and development but also activates the transcriptions in immune responses. So far, CBP60b has only been studied extensively in the model plant Arabidopsis and rarely in crops. In this study, Bean pod mottle virus (BPMV)-mediated gene silencing (BPMV-VIGS) was used to silence GmCBP60b.1/2 in soybean plants. The silencing of GmCBP60b.1/2 resulted in typical autoimmunity, such as dwarfism and enhanced resistance to both Soybean mosaic virus (SMV) and Pseudomonas syringae pv. glycinea (Psg). To further understand the roles of GmCBP60b in immunity and circumvent the recalcitrance of soybean transformation, we generated transgenic tobacco lines that overexpress GmCBP60b.1. The overexpression of GmCBP60b.1 also resulted in autoimmunity, including spontaneous cell death on the leaves, highly induced expression of PATHOGENESIS-RELATED (PR) genes, significantly elevated accumulation of defense hormone salicylic acid (SA), and significantly enhanced resistance to Pst DC3000 (Pseudomonas syrangae pv. tomato DC3000). The transient coexpression of a luciferase reporter gene driven by the promoter of soybean SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (GmSARD1) (ProGmSARD1::LUC), together with GmCBP60b.1 driven by the 35S promoter, led to the activation of the LUC reporter gene, suggesting that GmCBP60b.1 could bind to the core (A/T)AATT motifs within the promoter region of GmSARD1 and, thus, activate the expression of the LUC reporter. Taken together, our results indicate that GmCBP60b.1/2 play both positive and negative regulatory roles in immune responses. These results also suggest that the function of CBP60b is conserved across plant species.

Silencing GmBIR1 in Soybean Results in Activated Defense Responses.

Receptor-like kinases (RLKs) are a large group of pattern recognition receptors (PRRs) and play a critical role in recognizing pathogens, transducing defense signals, and mediating the activation of immune defense responses. Although extensively studied in the model plant Arabidopsis, studies of RLKs in crops, including soybean, are limited. When a BAK1-interacting receptor-like kinase (BIR1) homolog (referred to as GmBIR1 hereafter) was silenced by the BPMV (Bean pod mottle virus)-induced gene silencing (BPMV-VIGS), it resulted in phenotypes that were reminiscent of constitutively activated defense responses, including a significantly stunted stature with observable cell death on the leaves of the silenced plants. In addition, both SA and H2O2 were over-accumulated in the leaves of the GmBIR1-silenced plants. Consistent with this autoimmune phenotype, GmBIR1-silenced plants exhibited significantly enhanced resistance to both Pseudomonas syringae pv. glycinea (Psg) and Soybean mosaic virus (SMV), two different types of pathogens, compared to the vector control plants. Together, our results indicated that GmBIR1 is a negative regulator of immunity in soybean and the function of BIR1 homologs is conserved in different plant species.