Lijing Liu

The causative agent of anthracnose, Colletotrichum spp., is ranked among the top ten plant fungal pathogens due to its scientific and economic importance.1 Colletotrichum spp. can infect most plant...

Base editing is a powerful tool for artificial evolution to create allelic diversity and improve agronomic traits. However, the great evolutionary potential for every sgRNA target has been overlooked. And there is currently no high-throughput method for generating and characterizing as many changes in a single target as possible based on large mutant pools to permit rapid gene directed evolution in plants. In this study, we establish an efficient germline-specific evolution system to screen beneficial alleles in Arabidopsis which could be applied for crop improvement. This system is based on a strong egg cell-specific cytosine base editor and the large seed production of Arabidopsis, which enables each T1 plant with unedited wild type alleles to produce thousands of independent T2 mutant lines. It has the ability of creating a wide range of mutant lines, including those containing atypical base substitutions, and as well providing a space- and labor-saving way to store and screen the resulting mutant libraries. Using this system, we efficiently generate herbicide-resistant EPSPS, ALS, and HPPD variants that could be used in crop breeding. Here, we demonstrate the significant potential of base editing-mediated artificial evolution for each sgRNA target and devised an efficient system for conducting deep evolution to harness this potential.

Sorghum relies on its immune system to defend against various pathogens, including Pseudomonas syringae syringae van Hall (Pss van Hall). However, the sorghum immune system is largely unknown. Reports on pathogenic effectors have provided valuable insights into the plant immune system; thus, we aimed to identify Pss van Hall effectors that can regulate the sorghum defense response in this study. Here, we first established the sorghum-Pss van Hall pathosystem and found that type III effectors played critical roles in the virulence of Pss van Hall to sorghum. To predict its effectors, the whole genome of Pss van Hall was sequenced, and 18 effector-coding genes were identified. Among them, five effectors belong to the core effectors of Pseudomonas syringae pathovars, and two may be monocot pathogen-specific effectors. Pss van Hall triggered the hypersensitive response (HR) in Nicotiana benthamiana. We found that the effectors of Pss van Hall can be divided into cell death inducers and immune repressors by examining their functions in HR induction and repression of PTI marker gene, ROS production, and pathogen growth. Finally, the roles of core effectors HopAJ2 and HopAN1, and specific effector HopAX1 were further confirmed in the sorghum-Pss van Hall pathosystem. Importantly, the functions of HopAN1 and HopAX1 in regulating plant immunity were reported for the first time. We believe that the identification of these effectors will facilitate the continued exploration of the sorghum immune system.

SUMMARY: Sorghum is an important crop for food, forage, wine and biofuel production. To enhance its transformation efficiency without negative developmental by‐effects, we investigated the impact of GRF4‐GIF1 chimaera and GRF5 on sorghum transformation. Both GRF4‐GIF1 and GRF5 effectively improved the transformation efficiency of sorghum and accelerated the transformation process of sorghum to less than 2 months which was not observed when using BBM‐WUS. As agrobacterium effectors increase the ability of T‐DNA transfer into plant cells, we checked whether ternary vector system can additively enhance sorghum transformation. The combination of GRF4‐GIF1 with helper plasmid pVS1‐VIR2 achieved the highest transformation efficiency, reaching 38.28%, which is 7.71‐fold of the original method. Compared with BBM‐WUS, overexpressing GRF4‐GIF1 caused no noticeable growth defects in sorghum. We further developed a sorghum CRISPR/Cas9 gene‐editing tool based on this GRF4‐GIF1/ternary vector system, which achieved an average gene mutation efficiency of 41.36%, and null mutants were created in the T0 generation.

Plants produce a burst of reactive oxygen species (ROS) after pathogen infection to successfully activate immune responses. During pattern-triggered immunity (PTI), ROS are primarily generated by the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD). RBOHD is degraded in the resting state to avoid inappropriate ROS production; however, the enzyme mediating RBOHD degradation and how to prevent RBOHD degradation after pathogen infection is unclear. In this study, we identified an Arabidopsis (Arabidopsis thaliana) vacuole-localized papain-like cysteine protease, XYLEM CYSTEINE PEPTIDASE 1 (XCP1), and its inhibitor CYSTATIN 6 (CYS6). Pathogen-associated molecular pattern-induced ROS burst and resistance were enhanced in the xcp1 mutant but were compromised in the cys6 mutant, indicating that XCP1 and CYS6 oppositely regulate PTI responses. Genetic and biochemical analyses revealed that CYS6 interacts with XCP1 and depends on XCP1 to enhance PTI. Further experiments showed that XCP1 interacts with RBOHD and accelerates RBOHD degradation in a vacuole-mediated manner. CYS6 inhibited the protease activity of XCP1 toward RBOHD, which is critical for RBOHD accumulation upon pathogen infection. As CYS6, XCP1 and RBOHD are conserved in all plant species tested, our findings suggest the existence of a conserved strategy to precisely regulate ROS production under different conditions by modulating the stability of RBOHD.

Introduction: DNA integration is a key factor limiting the marketing of CRISPR/Cas9-mediated gene edited crops. Several strategies have been established to obtain transgene-free gene edited plants; however, these strategies are usually time-consuming, technically difficult, providing low mutagenesis efficiency, and/or including a narrow host range.Method: To overcome such issues, we established a paraquat resistant 1 (PAR1)-based positive screening (PARS) strategy, which achieved efficient screening of transgene-free gene edited plants.Results: With PARS, the screening efficiency of mutant increased by 2.81-fold on average, and approximately 10% of T1 plants selected via PARS were transgenefree. Moreover, heritable transgene-free mutations at target loci were identified in the T1 generation.Discussion: Based on the previous reports and our data, we know that paraquat is toxic to all green plants, PAR1 is conserved among all plant species tested, and the transient expression of Cas9 editor can produce transgene-free gene edited plants. Thus, we assume that the PARS strategy established here has the potential to be widely used to screen transgene-free mutants in various crops using diverse CRISPR/Cas9 delivery approaches.

Plant defense responses under unfavorable conditions are often associated with reduced growth. However, the mechanisms underlying the growth-defense tradeoff remain to be fully elucidated, especially at the transcriptional level. Here, we revealed a Cys2/His2-type zinc finger transcription factor, namely, ZAT18, which played dual roles in plant immunity and growth by oppositely regulating the signaling of defense- and growth-related hormones. ZAT18 was first identified as a salicylic acid (SA)-inducible gene and was required for plant responses to SA in this study. In addition, we observed that ZAT18 enhanced the plant immunity with growth penalties that may have been achieved by activating SA signaling and repressing auxin signaling. Further transcriptome analysis of the zat18 mutant showed that the biological pathways of defense-related hormones, including SA, ethylene and abscisic acid, were repressed and that the biological pathways of auxin and cytokinin, which are growth-related hormones, were activated by abolishing the function of ZAT18. The ZAT18-mediated regulation of hormone signaling was further confirmed using qRT-PCR. Our results explored a mechanism by which plants handle defense and growth at the transcriptional level under stress conditions.

SUMMARY: Viruses cause many severe diseases in both plants and animals, urging us to explore new antiviral strategies. In their natural reservoirs, viruses live and replicate while causing mild or no symptoms. Some animals, such as bats, are the predicted natural reservoir of multiple viruses, indicating that they possess broad‐spectrum antiviral capabilities. Mechanisms of host defenses against viruses are generally studied independently in plants and animals. In this article, we speculate that some antiviral strategies of natural reservoirs are conserved between kingdoms. To verify this hypothesis, we created null mutants of 10‐formyltetrahydrofolate synthetase (AtTHFS), an Arabidopsis thaliana homologue of methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1 (MTHFD1), which encodes a positive regulator of viral replication in bats. We found that disruption of AtTHFS enhanced plant resistance to three different types of plant viruses, including the tomato spotted wilt virus (TSWV), the cucumber mosaic virus (CMV) and the beet severe curly top virus (BSCTV). These results demonstrate a novel antiviral strategy for plant breeding. We further discuss the approaches used to identify and study natural reservoirs of plant viruses, especially those hosting many viruses, and highlight the possibility of discovering new antiviral strategies from them for plant molecular breeding and antiviral therapy.

Key Message Transcriptome analysis of SA sensitive and tolerant lines indicates that SA enhances anthracnose resistance in sorghum by upregulating the expression of some immune-related genes and pathways. Abstract Anthracnose caused by the hemibiotrophic pathogen Colletotrichum sublineolum is one of the most destructive diseases of sorghum, the fifth most important cereal crop in the world. Salicylic acid (SA) is a phytohormone essential for plant immunity; however, the role of SA in sorghum resistance to anthracnose has not been well explored. In this study, we found that Colletotrichum sublineolum infection induced the expression of SA-responsive genes and that exogenous SA enhanced resistance to anthracnose in the sorghum line BTx623. To rule out the possibility that SA triggers anthracnose resistance in sorghum by its direct toxic function on pathogen, an SA-tolerant line, WHEATLAND, was identified, and we found that SA treatment could not induce anthracnose resistance in WHEATLAND. Then, SA-induced transcriptome changes during Colletotrichum sublineolum infection in BTx623 and WHEATLAND were analyzed to explore the molecular mechanism of SA-triggered resistance. SA pretreatment regulated the expression of 2125 genes in BTx623 but only 524 genes in WHEATLAND during Colletotrichum sublineolum infection. The cutin, suberine and wax biosynthesis pathway involved in the plant immune response and the flavonoid biosynthesis pathway involved in anthracnose resistance were enriched in BTx623-specifically upregulated genes. Additionally, some immune-related genes, including multiple resistance genes, were differentially expressed in BTx623 and WHEATLAND. Taken together, our results revealed the mechanisms of SA-induced anthracnose resistance in sorghum at the transcriptional level and shed light on the possibility of enhancing sorghum resistance to anthracnose by activating the SA signaling pathway by molecular breeding.